JPS63107941A - Carcinostatic agent - Google Patents

Abstract

PURPOSE:To obtain a carcinostatic agent, by adding a compound having carcinostatic action and a growth factor, a peptide corresponding to part of the constituent component of the growth factor, a derivative or a salt thereof as active ingredients. CONSTITUTION:A carcinostatic agent containing a compound (e.g. alkylating agent such as cyclophosphamide, antimetabolite such as 5-fluorouracil, etc., antibiotic such as mitomycin, etc., platinum preparation) and a growth factor (e.g. epidermal growth factor such as hEGF, etc., insulin group, growth factor group derived from blood platelet, etc.), a peptide corresponding to part of the constituent component of the growth factor, a derivative or a salt thereof as active ingredients. The dosage form may be oral or parenteral and optionally may be blended with other drug. A dose is preferably 10ng-10mg/person/day. A carcinostatic agent effective for any of all cancers can be prepared by properly selecting each active component.

Description

DETAILED DESCRIPTION OF THE INVENTION [Background of the Invention] Technical Field The present invention relates to a novel anticancer agent. In more detail,
The present invention relates to a compound having an anticancer effect, a growth factor, and a peptide (fragment) corresponding to some of its constituent components.
, derivatives thereof, or salts thereof (hereinafter sometimes referred to as "growth factors, etc.") as active ingredients.

Prior art growth factor is ri
nvlv.

or 1n vttro, something that promotes the growth of animal cells and is not a nutritional substance (Ann
, Rev. Blochem, 45.531-5
58 (197f3)), conventional hormones and their carriers also fall into the category of growth factors.

Approximately 40 types of such factors are currently known, and the main growth factors include those classified into the insulin family (insulin, insulin-like growth factor (IGF-I),
, ICF-II, etc.), mammary gland stimulating factor (< M S F
＞), nerve growth factor <(NG F
GF)), transforming growth factors (TGF)
5TGFβ, TGF7, etc.), α Those classified into the platelet-derived growth factor family (platelet-derived growth factor (PDGF)), osteosarcoma-derived growth factor (
ODGF)), fibroblast growth factor (FGF)
)), etc.), others (colony-forming stimulating factor (CSF)
)), T cell growth factor, @tumor angiogenesis factor (T A
F )), DNA synthesis promoting factor ((D S F >)
, tumor-derived cell growth factors, fibroblast-derived growth factors (
F D G F )) etc.).

Among these growth factors, epidermal growth factor has been isolated from the urine of humans and horses as well as from the submandibular gland of rabbits, rats, and mice, and is present in mammalian animals across species. It is known that [Adv, Metab, Dl
s,, 8.265 (1975), Japanese Patent Publication No. 56-25
Publication No. 112, etc.]. Among them, human epidermal growth factor (
human Epidermal Growth Fa
ctor:hEGF) was developed in 1975 by Cohen (S.
[Proc, Natl, Aead, Sc1. t
] SA, 72. 1317 (19) 5)], and in the same year, Gregory H, Gregory) isolated human urogastrone (hu+lan urogastrone) from human urine, which has the effect of suppressing gastric acid secretion.
-UG) was introduced as (Nature, 25
7.325 (1975)) is the same substance as polypeptide, has a molecular weight of approximately 6000, is composed of 53 amino acid residues, and has three disulfide bonds in its molecule [Metabolism, 17.51- 58 (1980
)] (hereinafter, epidermal growth factor will be referred to as EGF).

The physiological and pharmacological activities of EGF that have been reported to date include its gastric acid secretion suppressing effect (Cut).
, Dan, 1877 (1975), 1did, 23
, 951 (1982)), anti-ulcer effect (Gut, 22
．． 927 (1981), Br1d, J. Surg.
, 64.830 (1977)], gastrointestinal mucosal protective effect [JP-A-60-9686], DNA synthesis promotion effect (Cut, 22.927 (1981), j.

Physiol, 325.35 (1982))
, corneal repair action [JP-A-59-65020], calcium release promoting action (Endocrlnology,
107.270 (1980)), wound healing promoting effect [PIast, Rlconstr, Surg.
84°788 (1979), J. Surg, Re.
s, 33.184 (1982)), anti-inflammatory action (Japanese Patent Application Laid-open No. 115784-1984), and analgesic action (Japanese Patent Application Laid-open No. 115785-1985).

By the way, growth factors are generally grown in vivo (r.
In vitro (abbreviated as J), it has been reported to have an oncogenic promoter effect under special conditions. For example, there is such a report regarding EGF, which is one of the active ingredients of the present invention (Surgic
al Forum 16.108 (1985), E
xperlentia 32 (7), 913-91
5 (197B), 5ctence201, 515-5
18 (197g), Cancer Res, 39.
239-243 (1979)).

On the other hand, in vitro (abbreviated as r in vltroJ), cancer cells that have receptors for growth factors,
It is well known that proliferation is actually suppressed by the addition of growth factors, and specifically, there is a report that the proliferation of cancer cells that have EGF receptors is rather suppressed by the addition of EGF ( J, Biol, Ch.
e+++,, 259, 7761-7786 (198
4), Toxicology Forum 9 (1), 55-
82 (198B)]. Also, if not suppressed, EG
There is also a report that addition of F alone does not promote cell proliferation at all (Int, J, Ce1l Clon
ing, 3.

407-414 (1985)].

On the other hand, "cancer" currently ranks as the leading cause of death in Japan, and various treatments are being used for this cancer. Cancer treatments include surgical therapy, radiotherapy, chemotherapy, etc., and the first two are local therapies. Local therapy is extremely difficult to apply when cancer has metastasized to areas other than the primary tumor, so in such cases, chemotherapy using anticancer drugs must be relied upon. However, the anticancer drugs used in this method usually have strong anticancer effects, but they also have strong side effects, and chemotherapy has its limits in this respect.

Currently, anticancer drugs used include alkylating agents (chitrodiene mustard-N-oxide, triethylene melamine, busulfan, carmustine, dacarbazine, etc.), antimetabolites (methotrexate, 6-
Mercaptopurine, 5-fluorouracil (5-FU
)), cytosine arapinoside (A r a -C)
), cyclocytidine, etc.), antibiotics (adriamycin (doxorubicin)), acanomycin C1 actinomycin D1 chromomycin A3, pleomycin, etc.), plant alkaloids (vincristine, demecoltine, etc.), prostaglandins, immunostimulants (polysaccharides such as Visivanino ρ and Krestin ■), platinum preparations (such as cisplatin), lymphokines, and monokines (such as interferon and interleukin-2).

As described above, there are many anticancer drugs currently in clinical use, but among them, there is a tendency for specific anticancer drugs to be used selectively for specific organ cancers.

Currently, cancers that can be survived or cured for 5 years with chemotherapy include childhood acute leukemia, Hodgkin's disease, Atamaru tumor, Ewing tumor, Wilms tumor, Burkitt's lymphoma, choriocarcinoma, and embryonal strangulation sarcoma. , skin cancer, ovarian cancer, breast cancer, multiple bone marrow tumors, neuroblastoma, etc.

However, most of these cancers are relatively rare, and no satisfactory anticancer drug has yet been developed for the most common cancers, such as stomach cancer, lung cancer (with some exceptions), liver cancer, esophageal cancer, and colon cancer. (Book “Oncology” p. 269 (1)
984) Nanzando).

Additionally, antimetabolites have other problems than those mentioned above. For example, 5-fluorouracil (A Colprehenslve Treatise), which is currently frequently used clinically.
.

5-13271PrQIILll Presss Ca
ncer ReS, 1181478 (1958), G
astroenterology, 48.430 (
1965), Cancer Treat, Rep.
, 62.583 (1987)) has one of the strongest anticancer effects, but it has side effects such as causing diarrhea and a decrease in white blood cells by acting on the gastrointestinal mucosa and bone marrow where cells are actively dividing. It has been reported (Pharmaco
lgical Pr1ciples of'Canc
er Treatment, 195 (1982))
It is known that the other anticancer drugs mentioned above also have various side effects [Illustrated Guide to Pharmacology JP 384-3]
85, Breakfast Bookstore (published in 1979)]. As described above, conventional anticancer drugs that have strong anticancer effects generally also have strong side effects. Furthermore, 1-(2-tetrahydrofuryl)-5-fluorouracil (tegafur) has relatively little toxicity and side effects, but is said to have somewhat inferior anticancer effects.

In general, drugs with fewer side effects also have weak anticancer effects.

In addition, many treatments have been carried out with the aim of enhancing the anticancer effect and reducing side effects by combining conventionally known anticancer drugs with various other anticancer drugs. However, no definitive treatment has yet been established.

Another drawback is that when an anticancer drug is continuously administered, the cancer may develop resistance to the drug, making it difficult to obtain an anticancer effect. This is a clinically extremely important and recently rapidly increasing problem. Multidrug therapy is an attempt to overcome drug resistance by combining multiple anticancer drugs with different mechanisms of action, but it cannot be said that the problem of resistance has been overcome, and the toxicity is higher than when using a single drug. is also much more serious.

Therefore, in order to solve these problems, it is desired to provide an anticancer agent that has a strong anticancer effect and has few side effects.

[Summary of the invention]

Summary The present invention aims to solve the above-mentioned problems, and as a result of extensive research, it has been found that when a compound with anticancer activity is combined with a growth factor, etc., the anticancer effect of the compound with anticancer activity is enhanced. We discovered that the anticancer effect was exerted even on cancers that were strengthened and resistant to anticancer drugs, and that the side effects of the compound were also reduced. Based on this knowledge, we completed a new anticancer drug. By providing this, the above objective is achieved.

Therefore, the anticancer agent according to the present invention has as active ingredients (a) a compound having an anticancer effect; and C) a growth factor, a peptide corresponding to a part of its constituent components, a derivative thereof, or a salt thereof; It is characterized by:

Naturally! The anticancer agent of the present invention contains the above-mentioned ingredients as active ingredients, and has the advantage of solving the above-mentioned problems, having a strong anticancer effect, and having few side effects.

Compounds conventionally used as anticancer agents or known as having anticancer effects have had various side effects as described above.

However, in the present invention, when a growth factor or the like is further added to the compound having an anticancer effect, this enhances the anticancer effect of the compound and also has an anticancer effect on cancers that are resistant to anticancer drugs. As mentioned above, this was done after confirming that the compound exhibits its own effects and also suppresses the side effects of the compound.

Therefore, the anticancer agent of the present invention will be useful as a drug for cancer treatment, particularly chemotherapy.

Incidentally, among the active ingredients of the drug of the present invention, growth factors and the like themselves have useful physiological and pharmacological activities. for example,
EGF, which is one of the growth factors, has a therapeutic effect on peptic ulcers when administered orally or parenterally (Japanese Patent Laid-Open No. 59-1
80270), gastrointestinal mucosal protective effect (Japanese Unexamined Patent Publication No. 1983)
-9686), wound healing promoting effect (the above Plas
t, Reconstr.

Surg, , the above-mentioned J, Surg, Res, ). Therefore, since growth factors and the like have such various effects, the drug of the present invention containing them as one of the active ingredients can be expected to have unique therapeutic effects.

[Detailed description of the invention]

The anticancer agent of the present invention contains the above-mentioned components (a) and (b) as active ingredients.

Compound with anticancer effect (ingredient (a)) In the present invention, the term "compound with anticancer effect" refers to a compound that can be used mainly for the treatment of cancer, and is used as an anticancer agent or an anti-malignant tumor agent. This refers to drugs that have cancer-fighting or anti-malignant tumor effects. The compound whose anticancer effect is to be enhanced by the present invention includes:
Any compound is included as long as it inherently has an anticancer effect, and examples of compounds that have already been confirmed to have this effect include the following.

1. Alkylating agents Examples include chloromethines, night fudien mustards, ethyleneiminos, alkylsulfonic acids, nitrosoureas, and epoxides.

[Botoko, "Cancer Chemotherapy jp 10-33 (1985) Published by Nankodo]" Here, in the present invention, "-" refers to any compound that shares a skeleton structure with these compounds and can have activity. The concept includes substituted or chemically modified derivatives and salts thereof.

Examples of such compounds include Nightfujiene Mustards, Iverit, Nightfujiene Mustard-N
-oxide, cyclophosphamide (e.g. JP-A-58-
41892, etc.), melphalan, chlorambucil, uracil mustard, dopane, alanine-MN, and the like. Ethyleneimines include triethylenemelamine, triethylenethiophosphoramide, carbazylquinone,
There are products such as Trenimon. Examples of nitrosoureas include carnistine and lomustine.

2. Antimetabolites such as folate antagonists (methotrexate, aminopterin, etc.), purine antagonists (6-mercaptopurine, 8-azaguanine, etc.), pyrimidine antagonists (5-mercaptopurine, 8-azaguanine, etc.)
Fluorouracil (for example, JP-A-55-7231, JP-A-51-65774, JP-A-52-48677, JP-A-52-42)
887, Publication No. 53-31676, etc.), tegafur, carmofur, etc.) [see the above-mentioned "Cancer Chemotherapy", pages 33 to 52]. Recently, NFT■ (UFT■), a mixed drug of tegafur and uracil, has been introduced.
) are also used clinically.

3. Antibiotics, such as adriamycins (Actinomycin > CSD
etc.), Azaserine, DON.

Sarcomycin (Sarkomyci II), Carzinophyllin (Carzinophyllin III), mitomycins (e.g., JP-A-60-67433, etc.), chromomycin 83, pleomycin (e.g., JP-A-60-67425, etc.), Pepromycin,
Daunorubicin (Daunorbici II), adriamycin (Doxorubicin (Doxorublci II)) which is an anthracycline antibiotic [for example, JP-A-60-178818, JP-A-60-67425, etc.], aclarubicin (Aclarubicl II)
), etc. [The above-mentioned bottom setting "Cancer therapy" p52-p77
reference〕.

4. Hormone drugs, such as sex hormones (testosterone derivatives, nisradiol derivatives, etc.), pituitary/adrenal cortical hormones (coccin, prednisone, dexamethacin, etc.)
[Refer to "Cancer Chemotherapy" above, pages 77-88]
.

5. Plant alkaloids demecolcine (DemecolclII), vinblastine (Vinblastlne), vincristine (
Vlncristine), podophyllotoxin (Po
dophyllotoxi II) etc.
Cancer Chemotherapy” p. 89-97].

66 Porphyrin substances include hematoporphyrin mercury complex salt, protoporphyrin cobalt complex salt, etc.
Reference] 76 Other immunostimulants (Krestin (PSK)
)),” bisibanil, lentinan, etc.), platinum preparations (
Cisplatin ((e.g., JP-A-56-152415, etc.), carpoplatin, ibroplatin, etc.)
14 (1986) Shinshu Igaku-sha], and other lymphokines and monokines (interferon, interleukins, etc.) [see the above-mentioned "Cancer Chemotherapy", pages 98-105].

Growth factors, etc. (ingredients (b)) In the present invention, "growth factors" refer to in vivo or 1n
As mentioned above, VLTR refers to substances that promote the growth of animal cells and are not nutritional substances, and conventional hormones and their carriers are also included in the category of growth factors.

As mentioned above, growth factors include the epidermal growth factor family,
There are insulin family, platelet-derived growth factor family, etc., all of which are subject to the present invention, but among these, representative ones are those of the epidermal growth factor family, such as epidermal growth factor (EGF) and transforming growth factor. factors (TGF) and insulin family members such as insulin and insulin-like growth factors (IGF-1, IGF-I
I) and members of the platelet-derived cell growth factor family, such as fibroblast growth factor (FGF).

The present invention provides not only the above-mentioned naturally occurring growth factors but also the arbitrary substitution of amino acids constituting the growth factors, addition or absence of amino acids, etc., which are different from the above-mentioned growth factors but similar to the growth factors. It also includes those having similar physiological and pharmacological activities, so-called fragments and derivatives. Note that the amino acid that can be arbitrarily substituted as a derivative may be any amino acid as long as it has the activity of a growth factor. (e.g., those with high fat solubility, etc.)]
It may be.

Further, the growth factor derivatives also include fragment derivatives. A fragment may be a portion that has at least binding activity to a growth factor receptor, and has a partial structure of a part of the growth factor. For example, one or more amino acids are missing from the C-terminus or N-terminus, or
It includes those formed by cleavage from the terminal or any arbitrary part and consists of two or more amino acids.

In addition, among the above factors and their derivatives mentioned above, derivatives that have not lost their terminal amino and carboxyl groups through chemical modification have free amino and carboxyl groups, so they cannot be used as salts with acids or with bases. It can also be salt. In that case, pharmaceutically acceptable organic or inorganic acids and bases can generally be used, such as hydrochloric acid, sulfuric acid, acetic acid, malonic acid, succinic acid, sodium hydroxide, amines, etc. can be mentioned.

Derivatives also include those subjected to various chemical modifications. Such derivatives are obtained, for example, by chemical modifications known per se, such as alkylation, oxidation, reduction, deglaciation, or combinations thereof.

Therefore, the present invention provides not only the above-mentioned growth factors themselves, but also
It also includes vegtides (fragments) having partial structures thereof, derivatives thereof, and salts thereof.

In addition, these growth factors etc. are used in living organisms (specifically, blood,
(Growth factors and their derivatives, etc. can be extracted from saliva, urine, lachrymal fluid, breast milk, various tissues/organs, etc.) or prepared by various methods [edited by the Japanese Society of Tissue Culture [Cell Growth Factor J]
(Refer to the literature included in Asakura Shoten, 1984)), chemical synthesis, or genetic engineering synthesis techniques. The growth factors etc. prepared in this way are preferably of high purity.

Epidermal cell growth factor is a typical example of such growth factors. As mentioned above, epidermal growth factor has been isolated from the urine of humans and horses, as well as from the submandibular glands of rabbits, rats, and mice, and is present in mammals across species. is known (Adv, Meta
b, Dls, 8.285 (1975), Japanese Patent Application Publication No. 1983
-25112, etc.]. Among these, when the drug of the present invention is applied to humans, the hEGF is preferred. For the preparation of this EGF, for example, a method of isolating it from biological components [JP-A No. 58-99418, No. 58-219124]
No. 59-204123, etc., Special Publication No. 1977-12
No. 744, No. 53-4527, No. 59-50315, No. 59-50316, No. 59-42650, etc.], chemical synthesis methods [such as Japanese Patent Application Laid-open No. 59-27858, etc.], and genetic chemistry methods. Method of creating by scientific method [
JP-A-57-122096, JP-A-58-216697, JP-A-59-132892, etc.] have been proposed.

The physiological and pharmacological activities of EGF that have been reported to date include its gastric acid secretion suppressing effect (Cut).
, 18.1887 (1975), 1bld, 23.
951 (1982)), anti-ulcer effect (Cut, 22.
927 (1981), Brlt.

J. Surg., 84, 830 (1977)), Protective effect on gastrointestinal mucosa [JP-A-9686], DN
A synthesis promotion effect (Cut, 22, 927 (19
81), J. Physiol, 325.35 (1
982)], corneal repair action [JP-A-59-65020
], Calcium release promotion effect (Endocrln)
ology, 107.270 (1980)], wound healing promoting effect [PIast, Reconstr, Surg.
,, 84.788 (1979), J. Surg, Re.
As mentioned above, it has anti-inflammatory effects (Japanese Unexamined Patent Publication No. 115784/1984), analgesic effects (Japanese Unexamined Patent Publication No. 115785/1983), etc.

On the other hand, EGF* conductors are those created by genetic engineering methods [one in which leucine is substituted for methineon at the 21st amino acid residue from the N-terminus among the 53 amino acid residues that constitute human EGF (hEGF) ((Leu21)- hEG
F: Japanese Unexamined Patent Publication No. 60-28994)] has been proposed. As another EGF derivative, hEGF-n, which is hEGF lacking two C-termini, has also been obtained [Patent application No. 60-2 proposed by co-researchers of the present inventors.
See specification of No. 2630. In addition, here, hEGF prepared by genetic engineering by the present inventors' co-researchers.
has high purity (approximately 99.9% or more), and such high purity is preferable as component (b). ]
. Yet another EGF fragment is des-(49-
53)-EGF (EGF-5), des-(48
-53) -EGF (EGF -6) (Bioc
h-emistry, 15.2824 (f97B)
, Mo1. Phara+aco1. , 17°314
(1980), Vitam, Horm, 37.89
(1979), Cl1n.

Res, 25.312A (1977)), EG
F is EGF-(20-31), which is considered to be the minimum unit for expressing its various effects, and its derivatives ((A
cm) 20.31 Cys ] EGF-(20-31) (Proc
.

Natl, Acad, Sc1. , 81, 1351. (1
984) and [Ala20]EGF-(14-31)
(Proe, Natl, Acad, Scf,.

81.1351 (1984)), CNBr, which is a derivative obtained by treatment with cyanide bromide.
-EGF (Bloche+*1stry, 15.
2B24 (197B) ] and the like, and such substances also fall within the scope of growth factors, etc. of the present invention.

One group of growth factors is the epidermal growth factor family, examples of which are the above-mentioned EGF and transforming growth factors.
Pac-tor (TGF) was mentioned above.

This TGF has large similarities in amino acid sequence with EGF, and is currently classified into three classes. That is, TGF.

α TGFβ and TGFa. TGFβ is TGF
Alternatively, it acts synergistically with EGF to promote cell α growth. TGF alone exerts this effect γ. The three types of TGF, EGF and this, belong to the same growth factor family, and TGF in particular has a high amino acid sequence similarity to EGFα, with up to 21 of the 50α residues of TGF being found at homologous positions in EGF. has been issued (Proc, Natl, Acad, Sc1..81
．． 7383 (19g4)]. Also, TGF is EGF
α binds to the receptor, which competes with EGF [“The History of Medicine” 1
33.1040-1044 (1985)].

Furthermore, TGF activates the EGF receptor kinase α, Nature, 292.259 (1981
)], anti-EGF receptor antibodies block the vulacrine action of TGF (J, Blol, Chem, 258.1189
5 (19B4)), from these facts, EGF and TG
It is understood that F has a very similar effect. Similarly, insulin and IGFs, TGF and EG
It has been reported that there is a similar effect, such as the relationship α with F (
Adv, Metab, Dis, 8.203,211
, 237 (1975), Endocrlnology,
107.1451 (1980)], and IGFs have growth factor activity in addition to insulin-like activity. The present invention also includes homologous factors having very similar effects as these.

Anticancer action regulating effect The effect of the present invention is that growth factors etc. enhance the original anticancer effect of a compound that has an anticancer action on a wide range of cancers, and (also) suppress or prevent the side effects of the anticancer drug. It consists of things.

Among these, the enhancement effect of the anticancer effect of the above drugs can be confirmed using the inhibition of cancer cell growth transplanted into experimental animals as an indicator when a growth factor is used in combination with a compound that has traditionally been used alone as an anticancer agent. can. In the present invention, a specific example thereof is mouse colon adenocarcinoma (C) caused by N-methyl-N-nitrosourethane.
olonadenocarcinoma 2B and C
The above-mentioned effects have been confirmed by transplanting human breast cancer, etc., into experimental animal mice, and examining the suppressive effect on cancer cell proliferation when the active ingredient of the drug of the present invention is administered.

Furthermore, the effect of suppressing or preventing side effects of the above compounds can be confirmed using the weight loss of experimental animals transplanted with cancer cells as an indicator. For details on confirming these effects, please refer to the experimental examples described later.

Anticancer Agent The anticancer agent according to the present invention contains the above-mentioned "compound having an anticancer effect" (a) and "growth factors, etc." (b) as active ingredients. Therefore, the anticancer agent of the present invention differs from conventional anticancer agents in that a growth factor and the like are added to the compound having an anticancer effect as another active ingredient.

The above-mentioned "compound having anticancer effect" in the formulation of the present invention
Since the blending ratio of (a) and "growth factors, etc." (b) differs depending on the type of compound with anticancer effect,
It may be determined as appropriate depending on each type.

For example, when (a) is 5-FU or tegafur and (b) is EGF, it is preferable to use (a) in excess of (b), with 10% of (a) per 1 mole of (b). ~10
About 8 moles, more preferably about 10 to 106 moles,
It is better to use

If (a) is adriamycin, mitomycin C, or cisplatin and (b) is EGF, insulin, TFG, or α IGF-11, (b) is 1 mole to (a) 1
It is preferable to use about 10' to 106 mol, more preferably about 10' to 106 mol.

When (a) is cyclophosphamide and (b) is EGF, insulin, TGF, or α is 5×
It is preferable to use 10 to 5×1055×10.

(A) is 5-FU or Tegafur and (B) is FCF
In the case of (b) 1 mole, (b) 10 to 109
It is preferable to use about mol, more preferably about 10 to 10 7 mol.

(a) is 5-FU or tegafur and (b) is EGF
and TGFβ, (b) 1 mol of ECF and TG
It is preferable to use (a) about 10 to 108 moles, more preferably about 10 to 106 moles, per 10 to 300 moles of Fβ.

When (a) is U-FT [F] and (b) is EGF, insulin, TGF, or EGFα-H, (a) 10 to 1 part by weight of (b)
It is preferable to use about 0.7 parts by weight, more preferably about 10 to 105 parts by weight.

The active ingredients (a) and (b) of the present invention may be formulated into various unit dosage forms and administered separately, or both ingredients may be pre-blended and administered as a mixture in various unit dosage forms. It may also be formulated into a dosage form and administered.

The timing of administration can be appropriately selected depending on the combination of the formulated active ingredients (a) and (b), and the latter may be administered at the same time as, or before or after, the former. In one embodiment of the invention, the antimetabolite 5-FU and hEGF
In cases such as combinations with hEGF (
It is preferable to administer the active ingredient (corresponding to (b)). In addition, the number of administrations also depends on the form of the drug and the active ingredient used.
Furthermore, it may be selected as appropriate depending on the patient's symptoms.

The anticancer agent according to the present invention usually consists of the above-mentioned active ingredient and auxiliary ingredients in the formulation. Among these, specific examples of auxiliary components include carriers (excipients, binders, diluents, etc.), stabilizers, preservatives, solubilizing agents, and the like. Examples of carriers include calcium carbonate, lactose, sucrose, sorbitol, mannitol, starch, amylopectin, cellulose derivatives, gelatin, cacao butter, water, paraffin, fats and oils, and the like. In the case of water, paraffin and fats and oils, in addition to acting as solvents, they may also form the anticancer agents of the invention in the form of emulsions or suspensions.

Various dosage forms can be selected depending on the therapeutic purpose, such as powders, granules, microgranules, tablets, pills, capsules, oral preparations such as troches, skewers, lotions, and injections. agent (for example, dissolving or suspending the active ingredient of the present invention in distilled water for injection, various infusion solutions, etc.),
In addition to parenteral preparations such as ointments and poultices, there may be any preparations that can be administered, such as those with sustained release properties or the like.

These can be administered orally or parenterally (including injections), and other drugs may be added as necessary.

The dosage may be determined depending on the age, weight, medical condition, etc. of the patient. For oral and parenteral administration (including injection), it is usually desirable to use about 10 ng to 10 mg of growth factors per day for adults. Note that there is no need to set any special conditions for the dosage of the compound having an anticancer effect, and it can be a known dosage. That is, 5-
For FU and Adriamycin, the daily injection dosage is on the order of 5-15a+g/kg body weight and 10-20 tng/kg body weight, respectively. Also, 5-F
The daily oral dosage for adults is 100 to 300
It is.

Preferred embodiments of the invention reduce this daily dose to 1
It is in unit dosage form for administration once or several times a day.

The anticancer agent according to the present invention injects the anticancer action regulator polypeptide into 5 male and female mice of group 97881 at a dose of 10 mg/kg by subcutaneous injection or by intravenous injection at a dose of IIIg/kg (each about 1 million times the human blood concentration). , equivalent to about 50,000 times the dose) and oral administration of 5 ml/kg), there was no change in general symptoms, and there were no deaths, indicating that the toxicity is low.

The cancer to be controlled by the anticancer agent of the present invention depends on the growth factors, etc. and the compound having anticancer effect among the active ingredients of the agent of the present invention. Therefore, the drug of the present invention can be prepared to be effective against all cancers by appropriately selecting the active ingredient (a) and the active ingredient (b).

EXPERIMENTAL FACTS The following experimental facts are intended to further specifically explain the present invention. The experimental examples relate to the regulation of anticancer effects, and the examples relate to formulations. These are examples, and the present invention is not limited thereto.

In addition, in experimental examples, when using compounds with anticancer effects and growth factors, etc., the same results were obtained whether they were injected individually (special order) or when they were injected together (mixed injection). Therefore, concomitant administration will be indicated in the separately ordered results. In addition, in accordance with the general rules for diurnal preparations: manufacturing method for injections in the Examples, for those that are not isotonic, the preparation may be strengthened as necessary.

Experimental Example 1 The anticancer effect of 5-FU, an antimetabolite, and hEGF was investigated in combination.

(1) Experimental animals BALB/c mice were kept in a constant temperature (23 ± 0.5°C), constant humidity (60 ± 5%) room for one week, and then mice weighing 18 to 20 i that seemed to be healthy were collected. A group of 6 to 12 animals was used in this experiment.

(2) Experimental method and results Co1on adenocarcinoaa 2B
(Mouse colon adenocarcinoma) minced into small pieces, about 0.1~
0.2 ml was subcutaneously transplanted into the right side of the mouse abdomen using a cell transplant needle (Trotsker). Six days after transplantation, the size of the cancer, that is, the major axis (amm) and the minor axis (bmm) of the cancer, was measured, and the weight of the cancer was calculated using the following formula [Instruction 14 (Instruction 14) (
MCI) 1980).

[This cancer mass generally takes the shape of a spheroid,
A formula for calculating the volume of a spheroid is introduced.

In addition, the specific gravity of cancer cells was assumed to be approximately 1, and the volume was defined as -ffl. Furthermore, at the beginning of the experiment, the cancer weight was measured immediately before administration of the compound, and the difference in the average cancer weight at the start of the experiment between each group was determined. Divide the mice into three groups using a random number table to prevent
After the average cancer weight of each group was equalized, one compound was administered to each group.

First, in order to examine the anticancer action enhancing effect of the present invention, a group in which 150 ag/kg of 5-FU, which has been conventionally frequently used as a compound with anticancer action, was administered subcutaneously alone;
FU, 150 mg/kg and human EGF (hEGF)1
Changes in the weight of transplanted cancer cells over time were measured in the subcutaneously administered group with 00 μg/kg and in the group without compound administration. The results obtained were as shown in FIG. In the figure, the horizontal axis shows the number of experimental days from the 18th compound administration port, and the vertical axis shows the cancer cell proliferation rate,
The weight of the gun at the 18th day of each group is taken as 100%, and the ratio of the weight of the gun after each number of days to this weight is taken as 100%.

■: hEGF + 5-FU combination administration group: 5-FU
Administration group ○: No compound administration group Kimoto: P<0.01 *: P<0.05 (*): P<0. 1 [However, P indicates the risk ratio when considering the statistical significance of the 5-FU monoadministration group, and these values are all based on the Student's T test (5student's test).
s t-test ) or a modified Aspln-Welch method. ] The test liquids to be tested are as follows.

hEGF: hEGF dissolved in a solvent (physiological saline containing 0.01% Tveen 80) to a concentration of 100 μg/10 ml/kg.

5-FU: 5-FU dissolved in a solvent (physiological saline containing 10% dimethyl sulfoxide) at a concentration of 150 tag/10 ml/kg.

In addition, to the control group (no compound administration group), both the hEGF and 5-FU solvents described above were administered.

As a result, in the group administered with 5-FU and hEGF in combination, cancer cell proliferation was not only suppressed compared to the group without compound administration, but also significantly more cancer cell proliferation than in the group administered with 5-FU alone. An inhibitory effect was observed, and the anticancer effect of 5-FU was significantly enhanced.

Experimental Example 2 Using adriamycin, an antibiotic anticancer drug,
The anticancer effect enhancement effect was investigated in the same manner as in Experimental Example 1 above.

Experimental methods and results The experiment was conducted in the same manner as in Experimental Example 1.

However, for the test solution administration group, Adriamycin 10mg/
Adriamycin 10 t.
A group received a combination of subcutaneous administration of trg/kg and 100 μg/kg of hEGF, and a group received no compound administration.

The results obtained were as shown in FIG.

The symbols in the figure are as follows.

hEGF + adriamycin combination administration group Δ Niadriamycin administration group, the rest is the same as above.

The test liquid was as follows.

hEGF: Same as Experimental Example 1.

Adriamycin Niadriacin■Note [Kyowa Hakko Kogyo■
] and use it as a solution by adding physiological saline to 10 mg/10 ml/kg.

From this result, adriamycin 10 mg/kg and hE
By using GF in combination with 100 μg/kg, a marked cancer growth suppressive effect was observed from the day after administration, and by 9 days after administration, cancer cells had decreased to approximately 3% compared to the adriamycin alone administration group.
The size was only about one-fold, and a statistically significant sustained anticancer effect enhancement effect was observed.

Experimental Example 3 Next, the anticancer effect enhancing effect was investigated in the same manner as in Experimental Example 1 using insulin among the growth factors.

Experimental methods and results The experiment was conducted in the same manner as in Experimental Example 1.

However, the test solution administration group was a group in which 5-FU 150 mg/abdominal was administered subcutaneously, a group in which 5-FU 150 mg/)cg and insulin 50 μg/rcg were subcutaneously administered in combination, and a group in which insulin 50 μg/kg was subcutaneously administered alone. and a compound-free group.

The results obtained were as shown in FIG. The symbols in the figure are as follows.

○: No compound administration group: 5-FU administration group ■: Insulin + 5-FU combination administration group Mouth: Insulin administration group The test solutions were as follows.

Insulin: Insulin dissolved in a solvent (physiological saline containing 0,005N hydrochloric acid) to give 50μg/10ml/kg 5-FU: Same as Experimental Example 1 From this result, insulin 50μg/)cg and 5-FU F0
When used in combination with 150 mg/kg, a remarkable cancer growth inhibiting effect was observed from the day after administration, and the size of the cancer was suppressed to approximately the same size as the injection port until 6 days after administration. 5-
Compared to administration of FU alone, this size was about half. As shown in the diagram (*), books, etc. are marked on the 5th floor.
A statistically significant sustained anticancer effect was observed compared to the group administered with U alone.

Experimental Example 4 (1) Experimental Animals C57B L/6 male mice were kept at a constant temperature (23±0.5°C).
After preliminary rearing for one week in a constant humidity (60±5%) room, 10 to 20 animals each weighing 18 to 20 g and considered to be healthy were used in the main experiment.

(2) Experimental Methods and Results Colon adenocare1noia38 (mouse colon adenocarcinoma) was minced and minced. 1~0
．． 2 ml was subcutaneously transplanted into the right side of the mouse abdomen using a cell transplant needle (Trotsker). Eleven days after transplantation, the size of the cancer, ie, the major axis (amm) and minor axis (b++m) of the cancer, was measured, and the cancer weight was calculated using the following formula in the same manner as Colon26.

[Since this cancer mass generally takes the shape of a spheroid, a formula for calculating the volume of a spheroid was introduced. In addition, the specific gravity of cancer cells was assumed to be approximately 1, and the volume-weight ratio was calculated. ] Before starting the experiment, immediately before administering the compound (11 days after cancer transplantation)
After one day), the cancer weight was measured, and the mice were divided into three groups using a random number table so that there would be no difference in the initial cancer weight between each administration group.After the average cancer weight of each group was equalized, each groups were administered the compound.

In order to examine the effect of enhancing the anticancer action of the growth factor according to the present invention, a group in which 150 μ/kg of 5-FU, which has been conventionally frequently used as a compound with anticancer action, was administered subcutaneously alone;
The above experiment was conducted on a group to which 150 μg/g of 5-FU and 100 μg/kg of hEGF were subcutaneously administered in combination, and a group to which no compound was administered. The results obtained were as shown in Table 1.

In the same table, the number of days indicates the number of days of the experiment, with the compound administration port being the 1st day and month, and the cancer weight immediately before compound administration on the 1st day and month is 100. Each numerical value represents the average Hanito standard error of 10 to 20 animals. In the table, (*), * and Kyo zero marks are 5-F
It shows a statistically significant difference between the U single administration group and the combination administration group (the definition of P is as above).

From this result, in the hEGF + 5-FU combination administration group,
From the 3rd day after administration, significant inhibition of cancer cell growth was observed in all groups compared to the 5-FU alone administration group. Also, hEG
(Leu2')-hEGF or hEGF instead of F
When the same experiment as above was conducted using -II, similar results were obtained.

J: I-I-1--He1ψLr'l-Ma[F]o Experimental Example 5
'hEGF, which is one of the growth factors, is involved in cancer production (
In order to examine the effect of reducing or suppressing the side effects of a compound having a function, the following measurements were performed in Experimental Example 1. (In other words, in Experimental Example 1, the value obtained by subtracting the cancer weight from the body weight of the experimental animal immediately before drug administration is 9100,
The ratio of the cancer weight to this weight was calculated by subtracting the gun weight from the total weight of the experimental animal on each day 2 after drug administration, and is shown in Figure 4. The symbol 2 in the figure is the same as in Experimental Example 1.

From this result, no suppressive effect on weight loss was observed when 5-FU was administered alone, but hEGF and 5-FU were not effective in suppressing weight loss.

A statistically significant weight loss suppressing effect was observed in the group administered in combination with -FU compared to the group without compound administration or the group administered with 5-FU alone. 1 Experimental Example 6 Using 5-FU, an anti-metabolite anticancer drug.

The anticancer effect of the combined use of hEGF and 5-FU on murine leukemic solid cancers resistant to 5-FU was tested by measuring the increase and decrease in the weight of the cells.

l) Experimental animals: CDF1 male and neutral mice. Breeding conditions, etc. are as per test example 1.
Same as .

:2> Experimental method and experimental results P2S5 (mouse leukemia) strain (P38815-FU) with drug resistance to 5-FU was pre-injected with CDF.
1 mice were grown intraperitoneally. Eight days after intraperitoneal transplantation, ascites fluid (containing cancer cells) was collected, diluted with liquid medium, and transferred to another mouse at 3 x 10 per mouse.
6 cells were subcutaneously transplanted into the axillary region of the mouse. Eight days after transplantation, the size of the cancer was measured in the same manner as in Experimental Example 1 (Colon 26), and the weight of the cancer was calculated.

The same operations as in Experimental Example 1 were performed for the groups of mice at the beginning of the experiment.

The administration of the compound was also carried out in the same manner as in Experimental Example 1. However, the number of administrations was twice, on the 1st and 3rd day.

The test liquids were as follows.

hEGF: Same as Experimental Example 1.

5-FU: Same as Experimental Example 1.

The results obtained are shown in Table 2.

In the same table, the number of days indicates the number of experimental days with the first compound administration port being the 1st day, and the gun weight immediately before compound administration on the 1st day is
It is set to 00. Each value represents the average Hanito standard error of 9 animals. In the table, (1), book and Hayashi marks indicate statistically significant differences between the 5-FU single administration group and the combination administration group (the definition of P is as described above).

Based on this result, the hEGF + 5-FU combination administration group
From the day after administration, significant cancer cell growth inhibition was observed in the 5-FU alone administration group and the compound-untreated group.

Therefore, since hEG'F was effective against not only colon cancer but also leukemia, it is easy to think that hEG'F would exhibit a remarkable anticancer effect against many cancers.

In addition, mouse leukemia P388 used in this experimental example
has resistance to 5-FU, and the fact that anticancer effects were observed against it suggests that it will also be effective against cancers that have developed drug resistance.

:I111-e Oel -■ I--e1◇1 H8[F] Experimental Example 7 In a derivative of hEGF, the 21st methionine (Met) from the N-terminus of the amino acid sequence was replaced with leucine (Leu) ( (Leu21)-hEGF and hEGF-n, which is a fragment lacking two amino acid residues on the C-terminal side of the amino acid sequence of hEGF, in the same experimental system as in Experimental Example 6 above. 5-F
The anticancer effects of the combined use of U and the combined use of hEGF-n and 5-FU were tested by measuring the increase or decrease in cancer fH1.

Although hEGF-II is a fragment of hEGF, its receptor binding ability and biological activity are similar to those of hEGF (J, Blol, Chem, 247).

7 [159 (1972)).

In the experiment, the test solution administration group was subcutaneously administered with 5-FUI50■/Dan alone, and 5-FU150mg/)cg and [L
eu21)-hEGF 100 μg/kg subcutaneously administered group, 5-FUI 50 mg/kg and hEGF-■
The same procedure as in Experimental Example 6 was conducted except that the group was subcutaneously administered with 100 μg/kg and the group was not treated with the compound.

The test liquid is as follows.

The results obtained were as shown in Table 3.

The symbols in the same table are the same as those explained in Table 2.

From this result, (Leu”] -hEGF+5-FU
In the combination administration group and the hEGF-II+5-FU combination administration group, similar to hEGF, a remarkable anticancer effect was observed against cancers resistant to 5-FU.

Table 3 2 147.4±15.1121.2±10.53 2
27.9±16.1232.7±15.94 268.
8±32.6280.0±31.792.0±4.8 lines 94.5±5.1*Lin 108.3±18.1 99.8± 4.8 Ling Lin 160.4±12.3 180.5±20.4'' Experimental Example 8 Using cisplatin, a platinum-based anticancer drug, the anticancer effect enhancement effect was investigated in the same manner as in Experimental Example 1.

In the experiment, the test solution administration group was given 5 mg/k of cisplatin.
g alone subcutaneously administered group, cisplatin 5/kg and h
The same procedure as in Experimental Example 1 was conducted except that the group was subcutaneously administered with 100 μg/kg of EGF and the group was not treated with the compound.

The results obtained were as shown in FIG.

The symbols in the figure are as follows.

-: Cisplatin administration group ■: hEGF+cisplatin administration group The rest is the same as above.

The test liquids were as follows.

hEGF: Same as Experimental Example 1.

Cisplatin A injection (Nippon Kayaku ■) 5mg/10m
The dose was 1/kg.

From this result, cisplatin 5■/kg and hEGF10
By using cisplatin in combination with 0 μg/kg, a marked cancer growth suppressive effect was observed from the day after administration, and by 6 days after administration, the cancer mass had grown to only about half the size of the cisplatin alone administration group and the compound-untreated group. However, a statistically significant sustained anticancer effect was observed.

Incidentally, cancers of the digestive system are generally insensitive to cisplatin. This experiment demonstrated that hEGF can convert gastrointestinal cancers that are originally cisplatin-insensitive to cisplatin-sensitive cancer.

Note that when an experiment similar to the above was conducted using hEGF-II instead of hEGF, similar results were obtained.

Experimental Example 9 Similarly to Experimental Example 4, cisplatin, a platinum-based anticancer drug, and hEGF were combined to treat mouse colon cancer colon.
The anticancer effect enhancing effect on adenocarclnoIIa38 was investigated.

The experimental method was the same as in Experimental Example 4, and the test administration group was the same as in Experimental Example 8.

The results obtained were as shown in FIG.

The symbols and test liquid in the figure are the same as those in Experimental Example 8.

These results show that not only Colon adenocarcinoIIa26, a murine colon cancer with a relatively fast proliferation rate, but also Colon adenocarcinom, which has a slow proliferation rate and is closer to human cancer in terms of proliferation rate.
There was also a marked effect on colon cancer in A38 mice from the day after administration.
The combined effect of EGF was observed, and the size of the cancer mass remained smaller than the size immediately before administration until 5 days after administration.
The size was approximately 273 in the group administered with cisplatin alone.
A moderate and statistically significant sustained anticancer effect was observed.

In addition, instead of hEGF, (Leu21:l-hE
When an experiment similar to the above was conducted using GF, similar results were obtained.

Experimental Example 10 Using adriamycin, an antibiotic anticancer drug,
Similar to Experimental Example 2 above, it is a derivative of hEGF (Leu
”)-hEGFO anticancer action enhancing effect was investigated.

In the experiment, Adriamycin was administered for 10 days as the test solution administration group.
/kg single subcutaneous administration group, and separately adriamycin 10a+g/kg and (Leu”)-hEGF10
The experiment was carried out in the same manner as in Experimental Example 2, except that there was a group in which 0 μg/kg was subcutaneously administered in combination with the compound, and a group in which no compound was administered.

The results obtained were as shown in FIG.

The symbols in the figure are as follows.

Mu: [Leu21]-hEGF + adriamycin combination administration group Δ Niadriamycin administration group The rest is the same as above.

From this result, adriamycin 10■/)cg and (L
By co-administering 100 μg/'kg of eu21)-hEGF, a remarkable cancer growth inhibiting effect was observed from the day after administration, and even 9 days after administration, the amount was only about 1.5 times that at the injection site. In contrast, adriamycin 1
When 0.0 kg/kg was administered subcutaneously alone, the concentration reached approximately 5 times that of the injection port 9 days after administration.
When coadministered with u''1]-hEGF, the effect was not strong.

In the group in which (Leu'')-hEGF and adriamycin were administered together, a statistically significant and sustained anticancer effect was observed on the cancer weight of the group in which adriamycin was administered alone.

Experimental Example 11 The effect of enhancing the anticancer action of hEGF on human breast cancer was investigated using adriamycin, an antibiotic anticancer agent.

The anticancer effect was tested by measuring the increase or decrease in cancer weight.

(1) Experimental Animals: BALB/e AJcl-nu male and female nude mice After preliminarily rearing for one week in a sterile clean rack maintained at constant temperature (23 ± 0.5°C) and constant humidity (60 ± 5%), they were healthy. A group of 8 animals with a weight of around 20 F was used in this experiment. The mice used in the subsequent experiments were raised in the above environment.

(2) Experimental method and results MX-i (human breast cancer) was cut into approximately 1 mm square pieces and subcutaneously transplanted into the right side of the axilla of a mouse using a cell transplant needle (Trotsker). 17 days after transplantation, measure the size of the cancer, that is, the diameter (amm) and short axis (bm + s) of the cancer,
Gun weight was calculated in the same manner as Co1on 26 using the following formula.

(This cancer M lump generally takes the shape of a spheroid, so
A formula for calculating the volume of a spheroid is introduced.

In addition, the specific gravity of cancer cells was assumed to be approximately 1, and the volume-weight ratio was calculated. ) To begin the experiment, immediately before compound administration (17 days after cancer transplantation)
After one day), the cancer weight was measured, and the mice were divided into three groups using a random number table so that there would be no difference in the initial cancer weight between each administration group.After the average cancer weight of each group was equalized, each groups were administered the compound.

In order to examine the anticancer action enhancing effect of the growth factor according to the present invention, a group in which 10 mg/kg of adriamycin, which has been conventionally frequently used as a compound with anticancer action, was administered subcutaneously alone, a group in which adriamycin 110 ll1/kg and hEGF
Gun weights were measured for the group to which 100 μtr/floor was administered in combination and the group to which no compound was administered. The results obtained were as shown in Table 4. In the same table, the number of days indicates the number of experimental days with the first compound administration port being the first day, and the gun weight immediately before compound administration on the first day is 100.

The compound was administered on the 1st, 8th, and 100th day133.
I went a total of 4 times each day. The test liquid was as follows.

hEGF: Same as Experimental Example 1.

Adriamycin: Same as Experimental Example 2.

Each value represents the average Hanito standard error of 8 animals.

The mark zero* in the table indicates that the cancer weight in the adriamycin-administered group was statistically significantly suppressed compared to the adriamycin-administered group (the definition of P is as described above).

From this result, significant and sustained suppression of cancer cell growth was observed in the hEGF + Adriamycin combination administration group from the 4th day after administration, compared to the Adriamycin alone administration group.

In other words, the combination of hEGF and anticancer drugs has been found to enhance the anticancer effect not only on experimental tumors in mice (also on human cancers).

Therefore, it is easy to imagine that the solution could be a beneficial drug for human cancer as well.

to! bu111(t) t -1(t)(t)(t)
Using cisplatin, each of these and hEGF, [Leu
21) When the same experiment as above was conducted regarding the effect of combined use with hEGF or hEGF-n, the same results as above were obtained.

Experimental Example 12 Using hEGF-II, which is a fragment lacking two C-terminal amino acid residues in the amino acid sequence of hEGF, the anticancer activity enhancing effect was investigated in the same manner as in Experimental Example 1 above.

Although hEGF-n is a fragment of hEGF, it has the same receptor binding ability and biological activity as hEGF, as described above.

Experimental Method and Results The experiment was conducted in the same manner as in Experimental Example 1. However, the test solution administration group was a group in which 5-FU150I1g/kg was administered subcutaneously alone, and a group in which 5-FU150mg/kg and hEGF-100
A group was administered subcutaneously in combination with μg/kx, and a compound administration group.

The results obtained were as shown in Table 5.

In the same table, the number of days refers to the number of experimental days with the compound administration port being the 10th day, and the can weight immediately before the 10th compound administration is 100 days.
It is said that Each value represents the mean value ± standard error of 6 to 12 animals. In the table, the marks (*), *, and *0 indicate a statistically significant difference between the 5-FU single administration group and the combination administration group (
The definition of P is as above).

From this result, significant and sustained cancer cell suppression was observed in the hEGF-II+5-FU combination administration group compared to the cancer weight of the 5-FU alone administration group from the next day after administration.

ocomtntobui-1 -t-+ + r-+ r+ r-t + [F]
a'l(t)m- Experimental Example 13 Using (Leu21)-hEGF, which is a hEGF derivative in which the 21st methionine from the N-terminus of the hEGF amino acid sequence was substituted with leucine (Leu), the experiment described in Experimental Example 1 was conducted. Similarly, the anticancer effect enhancement effect was investigated.

Experimental methods and results The experiment was conducted in the same manner as in Experimental Example 1.

However, the test administration groups were a group in which 5-FU150ag/kg was administered subcutaneously alone, a group in which 5-FU150a+g/kg and (L
A group was subcutaneously administered with 100 μg/kg of eu”-hEGF, and a group was not administered with the compound.

The results obtained were as shown in FIG.

The symbols in the figure are as follows.

O: Group without compound administration.

・;5-FU administration group.

■: [Leu2')-hEGF+5-FU combination administration group.

The test liquids were as follows.

(Leu21)-hEGF: 100μg/10ml/
(Leu”’)-hEGF was added to the solvent (0 kg).
, pH 7.4 physiological saline containing 01% Tween 80).

5-FU: Same as Experimental Example 1.

From this result, rLeu"')-hEGF100pg
By using 50mg/ksg and 5-FUI 50mg/)cg in combination, a remarkable cancer growth inhibiting effect was observed from the day after administration, and the size of the cancer was suppressed to approximately the same size as the injection port until 9 days after administration. Compared to the 5-FU alone administration group,
This size was approximately 174 even after 9 days. As indicated by (*), *, and zero* in the figure, a statistically significant enhancement of the sustained anticancer effect was observed compared to the 5-FU monoadministration group.

In addition, cyclophosphamide, an alkylating agent, was used instead of 5-FU, and this and hEGF or hEGF-n
When the same experiment as above was conducted for each combination, similar results were obtained.

Experimental Example 14 5-Fluorouracil (5-FU
) The anticancer effect enhancing effect was investigated in the same manner as in Experimental Example 4 using tegafur (futrafur), which changes into

In the experiment, the test solution administration group was given Tegafur 400II 1g/
tegafur 400 mg/k, group administered subcutaneously with 400 mg/k
The experiment was conducted in the same manner as in Experimental Example 4, except that a group in which 100 μg/kg of hEGF and 100 μg/kg of hEGF were administered subcutaneously and a group not treated with the compound were used.

The compound was administered twice in total, on the 1st day and the 4th day. The test liquid was as follows.

hEGF: Same as Experimental Example 1.

Tegafur: Futofur ■ Note (Colon Pharmaceutical Industry ■) 4
It was used at a dose of 00 mg/10 ml/kg.

The results obtained were as shown in Table 6.

In the same table, the number of days indicates the number of experimental days with the first compound administration port being the 1st day, and the gun weight immediately before compound administration on the 1st day is
It is set to 00.

Each value represents the mean value ± standard error of 7 animals. The marks in Table Chukyo and Kimoto indicate that the cancer weight in the combination administration group was statistically significantly suppressed compared to the tegafur administration group alone (the definition of P is as above).

From this result, significant and sustained suppression of cancer cell growth was observed in the hEGF + tegafur combination administration group from the next day after administration, compared to the tegafur alone administration group.

Also, instead of hEGF (Leu21)-hEG
When experiments similar to those described above were conducted using F or hEGF-II, similar results were obtained.

1. Experimental Example 15 Using cyclophosphamide, which is classified as an alkylating agent among anticancer agents, the anticancer action enhancing effect was investigated in the same manner as in Experimental Example 4. In the experiment, the liquid test administration group was subcutaneously administered with 200 mg/kg of cyclophosphamide alone, and the group with 200 mg/kg of cyclophosphamide and 100 μg of hEGF.
The experiment was carried out in the same manner as in Experimental Example 4, except that there was a group in which the compound was administered subcutaneously and a group in which the compound was not treated.

The test liquid was as follows.

hEGF: Same as Experimental Example 1.

Cyclophosphamide: Cyclophosphamide was dissolved in physiological saline at a concentration of 200wg/10ml/sample.

The results obtained were as shown in Table 7.

Compounds were administered the first time on day 1 and the second time on day 3. In the same table, the number of days indicates the number of experimental days with the first compound administration being the first day, and the gun weight immediately before compound administration on the first day is 100. Each value represents the average Hanito standard error of 8 animals. In the table, a book or a zero* mark indicates that the cancer weight in the combination administration group was statistically significantly suppressed compared to the cyclophosphamide alone administration group (the definition of P is as described above).

The results showed that in the hEGF + cyclophosphamide combination administration group, a significant and sustained cancer cell proliferation suppressive effect was observed from the next day after administration compared to the cyclophosphamide alone administration group.

to m--r-1r-1 -Qcn [F] Also, instead of hEGF (Leu213 -hEGF
When the same experiment as above was conducted using , similar results were obtained.

Experimental Example 16 Mitomycin C (MMC), an antibiotic anticancer drug
), the anticancer enhancing effect was investigated in the same manner as in Experimental Example 4.

In the experiment, the test administration group was a group in which MMC5tag/kg was administered subcutaneously alone, a group in which MMC5tng/kg and h
Group subcutaneously administered with EGF 100 μg/kg, MM
A group in which C5mg/kg and hEGF-I1100μg/kg were subcutaneously administered in combination, a group in which MMC5mg/kg and (Leu”
)-hEGF (100 μg/kg) was subcutaneously administered in combination with the group, and the compound was not treated.

The test liquid was as follows.

hEGF: Same as Experimental Example 1.

hEGF-n: Same as Experimental Example 8.

[Leu21]-hEGF: Same as Experimental Example 7.

MMC: MMC to be 5mg/10ml/kg
Dissolved in distilled water to make it isotonic with body fluids.

The results obtained are as shown in Table 8.

The compound was administered the first time on the first day and the second time on the fifth day. In the same table, the number of days indicates the number of experimental days with the first compound administration being the first day, and the gun weight immediately before compound administration on the first day is 100. Each value represents the standard error of the mean of 6 animals. In the table, the marks by Hon and Hayashi indicate that the cancer weight of each combined administration group was statistically significantly suppressed compared to the group administered with MMC alone (the definition of P is as described above).

From this result, hEGF + MMC, bEGF -n + M
A significant and sustained cancer cell proliferation suppressive effect was observed in the MC1(Leu'')-hEGF+MMC combination administration group from the next day after administration compared to the MMC alone administration group.

Table 8 2 194.9±58.1130.8±8.5973
239.8±57.6106.1±9.4964 28
2.5±15.5 95.8±5.5725 313.
6±21.398.6±3.9686 415.6±2
4.2160.7±10.9 907 524.5±5
0.6150.8±15.91008 590.6±6
1.5263.6±30.8130°9 603.3±
28.9308.7±54.0138°#
* , 2± 5.1 90.5±10.1 100.1
± 5.1*, 6± 5.7 98.3± 5.6
81.3±28.1.2±5.7*80.3±
9.2 85.6±29.1*
Hayashi, 6±10.5 54.4± 6.3 86
．． 3±14.3 Hayashi, 0±10.3 64.3±10.8 Hayashi 75.4
± 8.9#* *, 1± 6.7 98.8± 6.7 88.1
±9.6#0*, 3±30.3 144.4±28.1
160.3±15.8 lines, 9±21.8*156.
B±14.3*182.4±30.8 (*) Experimental Example 17 Mouse ovarian cancer M50 using platinum drug cisplatin
hEGF against 76 solid cancers.

[Leu''')-hEGF and hEGF-II, respectively, were tested for their anticancer effect enhancing effects when used in combination with cisplatin by measuring increases and decreases in cancer weight.

(1) Experimental Animals BDF 1 male mice were used. The rearing conditions were the same as in Experimental Example 1.

(2) Experimental Method and Results Mouse ovarian cancer M5076 was subcutaneously transplanted into the lower armpit of each mouse at 1 to 2×10 6 cells. After transplant 1
After 2 days, the size of the cancer was measured using Experimental Example 1 (Colon 26
), and the gun weight was calculated.

The same operations as in Experimental Example 1 were performed for the groups of mice at the start of the experiment. The compound was administered in the same manner as in Experimental Example 9.

The results obtained were as shown in Table 9.

The symbols in the table are as described above.

From this result, hEGF,
It can be said that it is clear that the combination of (Leu21)-hEGF, hEGF-■, and cisplatin further enhanced the anticancer effect of cisplatin alone. This effect was statistically significant and sustained.

Table 9 2144,9±15.9126.4±9.09≦315
3.9±14.9125.49±8.310!4194
．． 6±18.2136.9±5.210″:5207.
4±20.9143.6±14.511=6 22
5.2±19. 1 192. 0 ±20. 6
13ri), 1±6.7 lines 100.1±8.7(*)
95.3±4.8#5.2±13.2 95.6±8
．． 6*-98, 4±6.5*3.6±11.7*101
．． 1±5.6” ioo, 1±4.5”3.6±9
．． 4(*)120.3±10.1 115.6±6.
3(*)5.4±14.7*140.6±24.5
144.4±10.8*Also, mitomycin C, an antibiotic, was used instead of cisplatin, and this and hE
GF.

(Leu") - When the same experiment as above was conducted using hEGF or hEGF-II, the same results as above were obtained.

Experimental Example 18 Similar to Experimental Example 4, hEGF, (Leu21)-hEG
F, hEGF-n, and 5-FU were used to examine the anticancer effect enhancing effect on solid mouse ovarian cancer M5076 in the same manner as in Experimental Example 17.

The experiment was conducted in the same manner as in Experimental Example 1.7 for the test liquid and in Experimental Example 15 for the others.

The results were as shown in Table 10. The symbols in the table are as described above.

1'' Table 10 2 144.9±15.9114.7±7.5100.
93 153.9±14.9115.2±8.3 93
．． 84 194.6±18.2134.6±8.9 9
3.95 207.4±20.9187.3±19.3
123.96 225.2±19.1230.1±29
．． 4140.1± 7.9 98.6± 9.8
101.3± 8.6" ±4.8 100.1±5.6 89°5±11
．． 3(*)±9.2#95.9±8.3” 90.
3±10.1”±14.1” 116.5±20.3
134.4±9.8 books* ±11.8 166.4±21.4(*)155.5
±15.0 cells These results indicate that the combination of hEGF, (Leu'')-hEGF, and hEGF-■ with 5-FU further enhanced the anticancer effect of 5-FU alone against ovarian cancer in mice. is clear.

This effect was statistically significant and sustained.

Furthermore, when we conducted the same experiment as above using tegafur instead of 5-FU, which is an antimetabolite, and hEGF, (Leu21)-hEGF, or hEGF-n, we obtained the same results as above. Obtained. Furthermore, similar results were obtained using the alkylating agent cyclophosphamide.

Experimental Example 19 Using fibroblast growth factor (FGF), which is classified into the platelet-derived growth factor family among growth factors,
Similarly, the anticancer effect was investigated.

Experimental Method and Results The experiment was conducted in the same manner as in Experimental Example 4. However, hEGF was changed to FGF.

The test liquid is as follows.

FGF: FGF (Toyobo) was dissolved in physiological saline containing 0.001% Tween 80 to a concentration of 100μg/10ml/kg.

5-FU: Same as Experimental Example 1.

The results obtained are as shown in Table 11.

Note that the descriptions in the same table are the same as those in the experimental example above. Each number is 6-8
Mean values ± standard error of animals are shown. Statistically significant differences, etc. are the same as in the above experimental example.

From this result, the FGF + 5-FU combination administration group had a statistically significant effect on the 5-FU alone administration group from the next day after administration.
Moreover, a sustained cancer growth inhibiting effect was observed.

In addition, instead of 5-FU, the antibiotic mitomycin C, the alkylating agent cyclophosphamide, and the platinum drug cisplatin are used. When the same experiment as above was carried out using this method, the same results as above were obtained.

Experimental Example 20 Human IGF-n (
The anticancer effect of the combined use of hlGF-II) and the following compounds with anticancer activity was examined by measuring the increase/decrease in cancer weight.

The target compounds with anticancer activity are as follows. Tegafur: 400mg/dizziness, subcutaneous administration 5-
FU: 150+mg/kg - subcutaneously administered mitomycin-C: 5a+g/kg, subcutaneously administered cisplatin: 5■/kg, subcutaneously administered In the experiment, the administration group was divided into a group in which the above compound was administered alone at the indicated dosage, and a control group. Compound anastomosis and hIGF-II with cancer effects
The same procedure as in Experimental Example 4 was conducted except that the group was subcutaneously administered with 50 μg/ksr and the group was not treated with the compound.

The test liquid is as follows.

hIGF-n: hlGF-n was dissolved in a solvent (1/15M sodium phosphate buffer at pH 7.7 containing 0,001% Tveen 80) to give a concentration of 50 μg/10 ml/)cg.

Tegafur: Same as Experimental Example 14.

5-FU: Same as Experimental Example 1.

Mitomycin-C: Same as Experimental Example 16.

Cisplatin: Same as Experimental Example 8.

The results obtained were as shown in Table 12.

The numbers in the same table are based on the cancer weight immediately before compound administration.
The average value ± standard error of 8 to 10 animals is shown. In the table, the marks (*), *, and ** indicate a statistically significant difference between the group administered with IGF-■ and the group administered alone with each compound anastomosis having an anticancer effect (the definition of P is as described above). street). still,
This result is the value 4 days after administration of the compound.

From this result, in the group administered in combination with IGF-II and the above-mentioned compound having an anticancer effect, remarkable suppression of cancer cell proliferation was observed compared to the group administered with each compound having an anticancer effect alone.

Table 12 Administration group Cancer weight ratio (%) Compound untreated group 205.1
±12.3 tegafur single administration group
95.2±15.4 IGF-11 + Tegaflu combination administration group 49.8± 9.5*5-FU alone administration group 84.5± 7.0
ICF-I [+5-FU■ combination administration group 50
．． 6±4.8#Mitomycin-C single administration group
95.8± 5,51GF-11 + Mitomycin-C combination administration group 73.4± 5.8 Cisplatin single administration group 145.6±
9. IIGF-n + cisplatin combination administration group
98.4± 8.2 # Experimental Example 21 Using insulin, in the same experimental system as in Experimental Example 4,
The anticancer effect of the combined use of insulin and the following compounds having anticancer activity was tested in the same manner as in Experimental Example 20.

The compounds with anticancer effect and the test liquid are as follows.

Tegafur: 400mg/kg, subcutaneous administration,
Same as Experimental Example 14.

Mitomycin-C: 5 mg/kg, subcutaneous administration, same as Experimental Example 16.

Adriamycin: 10 mg/kg, subcutaneous administration, same as Experimental Example 10.

Cisplatin: 5 ng/kg, subcutaneous administration, same as Experimental Example 8.

Insulin = 50 μg/kg, subcutaneous administration, same as Experimental Example 3.

The results obtained were as shown in Table 13.

The numerical values and symbols in the table were the same as in Experimental Example 23.

From these results, the group administered the combination of insulin and the above-mentioned compound having anti-cancer action had a more pronounced anti-cancer effect than the group administered alone with all the above-mentioned compounds having anti-cancer action.

Table 13 Administration group Cancer weight ratio (%) Compound untreated group 205°1±
12.3 Tegafl alone administration group
95,2 Sat 15,4 Insu 1 + Tegaflu combination administration group 56.3± 3,2*Mitomycin-
C alone administration group 95.8± 5.5 Insulin + mitomycin-C combination administration group 70.6±
4,3# Adriamycin single administration group
102.4 ± 10.4 Insulin + Adriamycin combination administration group 70.2 ± 6,5 * Cisplatin alone administration group 145.6 ± 9.1 Insulin + Cisplatin combination administration group 90.6
± 5.8 # Experimental Example 22 Using human transforming growth factor α (hTGF) α among transforming growth factors, hTGF was tested in the same experimental system as in Experimental Example 4 above.
The anticancer effect of α in combination with the following compound having an anticancer effect was examined by measuring the increase/decrease in cancer weight.

The experiment was an experimental example, except that the administration groups were a group in which each of the following compounds with anticancer activity was administered alone, a group in which insulin and the following compound anastomosis with anticancer activity were administered in combination, and a group not treated with the compound. It was carried out in the same manner as in 20.

The test liquid is as follows.

hTGF: TGF was dissolved in a 1715M sodium phosphate buffer at pH 5.9 containing α and 0.001% Tveen 80 to give 100μg/10ml/kg.

Compounds with anticancer effects: 5-FU: Same as Experimental Example 1.

Tegafur: Same as Experimental Example 14.

Mitomycin-〇2 Same as Experimental Example 16 Adriamycin: Same as Experimental Example 10.

Cisplatin: Same as Experimental Example 3.

The results obtained were as shown in Table 14.

The symbols in the table are the same as those in Table 12.

From this result, TGF α has various anticancer effects. There was a significant anticancer effect in the group administered with the same compound.

Table 14 Administration group Cancer weight ratio (%) Compound untreated group 205.1±
12.35-FU single administration group
84.5± 7.0 hTGF + 5-FU combination administration group 42.3 ± 5.3 #α Tegafur alone administration group 95.
2±15.4 hTGF + tegafur combination administration group
38,6±8,9#α Mitomycin-C alone administration group 95.
8±5.5 hTGF + mitomycin-〇 combination administration group 69.8± 7.8neα Adriamycin alone administration group 102.
4±10.4hTGF + Adriamycin combination administration group 63.8±8.9neα Cisplatin single administration group 145.
6± 9.1 Experimental Example 23 Using hTGFβ and hEGF, in the same experimental system as in Experimental Example 4, the anticancer effect of the combination of hTGFβ + hEGF and the following compound with anticancer effect was measured by measuring the increase or decrease in cancer weight. It was verified by

In the experiment, the test solution administration group was subcutaneously administered with the following compound having an anticancer effect alone, and the following compound with an anticancer effect plus hTGFβ 50 vag/kg and hEGF 1
The same procedure as in Experimental Example 4 was conducted except that the group administered 00 μg/kg in combination with the compound and the group not treated with the compound were used.

The test liquid is as follows.

hTGFβ: hTGFβ (871 Company) was dissolved in physiological saline containing 0.001% Tween 80 to a concentration of 50mg/10ml/kg.

hEGF: Same as Experimental Example 1.

Compounds having anticancer effects are as follows.

5-FU: Same as Experimental Example 1.

Tegafur: Same as Experimental Example 14.

Mitomycin-C: Same as Experimental Example 16.

Adriamycin: Same as Experimental Example 1O.

Cisplatin: Same as Experimental Example 3.

Cyclophosphamide: Same as Experimental Example 15.

The results obtained were as shown in Table 15. The symbols in the table are the same as those in Table 12.

Note that this result was obtained on the 4th day after administration of the compound.

From this result, a remarkable anticancer effect was obtained in the group administered with the combination of hTGFβ+hEGF and each compound having an anticancer effect.

Table 15 Administration group Cancer weight (%) Compound untreated group 216.3±20.
45-FU single administration group 1
03.3f15.4 Tegaflu single administration group
118.0± 9.8 mitomycin-
C single administration group 101.1±8.3
Adriamycin alone administration group 12
0.3±11,3 cisplatin single administration group
139.8± 9.9 Cyclophosphamide alone administration group 90.6± 7.6
Experimental Example 24 Using FGF, the anticancer effect of the combined use of FGF and tegafur was tested in the same experimental system as in Experimental Example 4 by measuring the increase/decrease in cancer weight.

The experiment was almost the same as Experimental Example 19. However, 5-F
I changed U to Tegafur.

The results were as shown in Table 16.

From this result, a statistically significant anticancer effect was observed based on the cancer weight of the FGF + tegafur combination administration group on the 4th day after administration.

Table 16 Administration group Cancer weight ratio (%) Compound untreated group 189.6 ± 9.8 Tegaflu single administration group
103.1±13.1 Experimental Example 25 hEGF%hEGF-n, (Leu21)-hEGF or Retinoic acid
id ) (RA) in the same experimental system as in Experimental Example 4, the anticancer effect of the combined use of these and tegafur was tested by measuring the increase/decrease in gun weight.

In the experiment, the test solution administration group was subcutaneous administration of tegafur 400 mg/kg alone, and the group administered tegafur 400 mg/kg and hEG.
F, hEGF-II, or (Leu21)-hEGF
The same procedure as in Experimental Example 4 was conducted, except that a group received subcutaneous administration of a combination of 100 μg/)cg and RA 30+ng/dazzle, and a group not treated with the compound.

The test liquid is as follows.

hEGF: Same as Experimental Example 1.

hEGF-n: Same as Experimental Example 7.

(Leu21)-hEGF: Same as Experimental Example 13.

RA: R to be 30mg/10ml/kg
A dissolved in olive oil.

Tegafur: Same as Experimental Example 14.

The results obtained are shown in Table 17.

The symbols in the same table are the same as those explained in Table 1. Note that this result was obtained on the 4th day after administration of the compound.

From this result, hEGF, hEGF-II, [Leu2
1] - A remarkable anticancer effect was observed in the group administered with tegafur in combination with hEGF and/or RA.

Table 17 Tegafur alone administration group 9
0.6±11.5h EGF + Theca7-/I, combination administration group 44. Of: 12.3”h
EGF-n + tegafur combination administration group 38
．． 6± 8.9#[Leu”']-hEGF+tegafur combination administration group 40.1± 7.4#hEGF+
RA+Tega7-Jl/combination administration group 20.3
Sat 5.8”hEGF-II+RA+Tegafur combination administration group 28.6±6.4#(Leu21)-h
EGF+RA+Tegafur 24.1±10. '
1” Experimental Example 26 NFT (U), a combination drug of tegafur and uracil
Similar to Experimental Example 4, the anticancer effect when used in combination with growth factors etc. was investigated using FT).

In the experiment, the test solution administration group was a group administered with UFT 650mg/kg, a group administered with a combination of UFT 650mg/kg (oral administration) and the following growth factors, etc. (subcutaneous administration), and a group not treated with compounds. The same procedure as in Experimental Example 4 was carried out except for the following.

The test liquid is as follows.

hEGF: Same as Experimental Example 1.

hEGF-n: Same as Experimental Example 7.

(Leu2')-hEGF: Same as Experimental Example 13.

hTGF: Same as Experimental Example 22.

α hTGFβ: Same as Experimental Example 23.

FGF: Same as Experimental Example 19.

Insulin: Same as Experimental Example 3.

hIGF-II: Same as Experimental Example 20.

UFT: The contents of a UFT capsule (Colorful Pharmaceutical Industry ■) was suspended in a 5% aqueous gum arabic solution and orally administered at a concentration of 650 mg/kg as UFT.

The results obtained were as shown in Table 18.

The symbols in the table are the same as those in Table 12.

From this result, it was found that the combined administration of growth factors etc. and UFT had a remarkable cancer growth inhibiting effect. The values in Table 18 indicate the cancer weight ratios on the 2nd day after administration of the compound, with the value immediately before administration of the compound taken as 100.

Table 18 Administration group Cancer weight ratio (%) Compound untreated group 110.3±
8.7UFT alone administration group 8
0.5±7.5hEGF+UFT combination administration group
51.7± 7.9 hEGF-11+UFT
Combination administration group 55.3±6.8 bottles (Leu
21) +UFT combination administration group 54.6±
8.1*hTGF + UFT combination administration group
40,3±5.1#α hEGF+hTGF+UFT combination administration group 41,
2± 6.5#β FGF+UFT combination administration group 59.9
± 5.1 icisulin + UFT combination administration group
58.6±s, 3(*)hIGF-n+UF
T combination administration group 53.8 ± 6.5 * Experimental Example 27 hEGF, hEGF-n and (Leu”) against solid cancer of murine leukemia resistant to adriamycin, an anthracycline anticancer antibiotic.
The anticancer effect of hEGF in combination with adriamycin,
The test was performed by measuring the increase or decrease in gun weight.

The experiment was conducted in substantially the same manner as in Experimental Example 5. However, the cancer cells were changed to P388/Adr, and the compound with anticancer activity was changed to Adriamycin.

The test liquids were as follows.

hEGF: Same as Experimental Example 1.

hEGF-II: Same as Experimental Example 7.

(Leu”) -hEGF: Same as Experimental Example 13.

Adriamycin: Same as Experimental Example 2.

The results obtained were as shown in Table 19.

The symbols in the same table are the same as those explained in Table 2.

From this result, anticancer effects were observed not only against 5-FU-resistant cancers but also against adriamycin-resistant cancers.

Therefore, it is thought that growth factors and the like will have a very effective effect on cancers that have developed drug resistance. still,
The values in Table 19 are the values on the 2nd day and month of administration of the compound, with the value immediately before administration taken as 100.

Table 19 Administration group cancer weight ratio (%)
No compound treatment group 181.
3±8.9 Adriamycin alone administration group
1.79.1±5.8hEGF+Adriamycin combination administration group 118.3±6.5#hEGF-n+Adriamycin combination administration group 120.5±5.9#(L
eu21)-hEGF+adriamycin 119.4
±8.5# Experimental Example 28 For the purpose of setting the duration of action of hEGF and the administration schedule, the sustainability of hEGF's anticancer action enhancing effect was investigated using 5-FU in the same manner as in Experimental Example 4. The experiment consisted of 5-F
A group received subcutaneous administration of U alone, and a group received subcutaneous administration of 5-FU and hEGF in combination. Among these, 5-FU and hEGF
The following six groups were established for the subcutaneous administration group.

That is, a group where hEGF was administered 3 days before administering 5-FU, a group where hEGF was administered 2 days before administering 5-FU, a group where hEGF was administered 1 day before administering 5-FU, a group where hEGF was administered 1 day before administering 5-FU, and a group where hEGF was administered 1 day before administering 5-FU. and hEGF co-administered group, 5-FU
A group in which hEGF was administered one day after administration of 5-FU, and a group in which hEGF was administered two days after administration of 5-FU. Other than that, the same procedure as in Experimental Example 4 was carried out.

The results were as shown in Table 20. Note that this result was measured 40 days after administering 5-FU.

From these results, we found that not only when 5-FU and hEGF were administered simultaneously, but also when hEGGF was administered 1 day before administering 5-FU.
Even with pre-administration of F, a remarkable suppressive effect on cancer growth was observed.

This was a completely unexpected event based on the tnvttro experiment, and indicates the long action time of hEGF.

Based on these results, a method of releasing hEGF in a sustained manner in vivo while injecting a compound having an anticancer effect such as 5-FU may be considered, and the range of possible applications may be greatly expanded.

Experimental Example 29 The following experiment was conducted regarding the usage of the compound (a) having anticancer activity and the growth factor (b) of the present invention.

That is, when administering (a) and (b) in combination, we attempted a method in which they were mixed and administered as a solution. The experimental method was the same as in Experimental Example 4.

The test liquid was administered in the same manner as in Experimental Example 4, except that (a) and (b) were co-injected only when they were used together.

(a) Tegafur: Same as Experimental Example 14.

Adriamycin: Same as Experimental Example 10.

Cisplatin: Same as Experimental Example 8.

(b) hEGF: Same as Experimental Example 1.

(Leu2')-hEGF: Same as Experimental Example 13.

hEGF-II; Same as Experimental Example 7.

TGF: Same as Experimental Example 22.

α Insulin: Same as Experimental Example 3.

FGF: Same as Experimental Example 19.

This result shows that mixed injection was able to achieve the same effect as custom injection.

This fact may be of pharmaceutical benefit.

Example 1) Human EGF for injection was dissolved in pyrogen-free 1715M phosphate buffer containing 0.1% w/v gelatin to a final concentration of 2.
The concentration was set to 0 μg/ml. For example, in the case of pH 7.4, sodium chloride was added at a final concentration of 75 mM. 5 ml of this solution was dispensed into each ampoule by sterile membrane filtration, for example, through a 0.22 μm Millex GV (Mlllex is a registered trademark) filter (manufactured by Millipore) and sealed.

Example 2) Human EGF for injection was dissolved in a pyrogen-free 1715M phosphate buffer containing 0.001 w/v% polyoxyethylene sorbitan fatty acid ester (polysorbate 80).
The final concentration was 20 μg/ml.

For example, in the case of pH 7.4, sodium chloride was added at a final concentration of 75 mM. This solution was sterilized and filtered in the same manner as in Example 1, and 5 ml each was dispensed into ampoules and sealed.

Example 3) Freeze-dried injection Mannitol is dissolved in the injection prepared in Example 1 above to a final concentration of 2 w/v%.

After this liquid was filtered for sterilization 2 in the same manner as in Example 1,
Dispense ml into glass vials and incubate at -40°C for 1
Freeze for an hour, -10℃, vacuum level 0.04m+sHg
The mixture was freeze-dried using a freeze dryer and sealed in a sterile manner using a conventional method.

Example 4) Freeze-dried injection A freeze-dried injection was prepared from the injection prepared in Example 2 in the same manner as in Example 3.

Example 5) Injection Human EGF 100μg Gelatin 5mg 5-Fluorouracil 1100II1 Trishydroxymethylaminomethane 400tng Distilled water for injection Appropriate amount (adjust to pH 8.0 with hydrochloric acid) 5ml per ampoule Same procedure as Example 1 with the above mixing ratio The mixture was sterilized and filtered, and an injection was prepared.

Example 6) Injection Human EGF 100 μg (Polysorbate 80) 50 μg 5-Fluorouracil 100 mg Trishydroxymethylaminomethane 400+ag Distilled water for injection Appropriate amount (Adjust to pH 8.0 with hydrochloric acid) 5 ml per ampoule With the above blending ratio, Example 1 and Sterilize and filter in the same manner,
An injection was prepared.

Example 7) Freeze-dried injection The injection prepared in Example 5 was dispensed into glass vials in 5 ml portions and frozen at -40°C for 1 hour.
0℃, degree of vacuum 0. The mixture was freeze-dried using a freeze dryer at O4 mmHg and sealed in a sterile manner using a conventional method.

Example 8) Freeze-dried injection From the injection prepared in Example 6, a freeze-dried injection was prepared in the same manner as in Example 7.

Example 9) Freeze-dried injection human EGF was dissolved in pyrogen-free physiological saline containing 0.1 w/v % gelatin to a final concentration of 20 μg/v.
The volume was adjusted to ml. Adriamycin hydrochloride was dissolved in this solution to a final concentration of 2/ml.

This solution was sterilized and filtered in the same manner as in Example 1, and a lyophilized injection was prepared using the same procedure as in Example 7.

Example 10) Lyophilized injection human EGF was dissolved in pyrogen-free physiological saline containing 0.001 w/v% polyoxyethylene sorbitan fatty acid ester (polysorbate 80) to a final concentration of 20μ.
g/ml. Adriamycin hydrochloride was dissolved in this solution to a final concentration of 2 mg/ml. This solution was sterilized and filtered in the same manner as in Example 1, and a lyophilized injection was prepared using the same procedure as in Example 7.

Example 11) Lyophilized injection Human EGF was dissolved in pyrogen-free physiological saline containing 0.1 w/v% gelatin to a final concentration of 20 μg/m.
It was prepared to l. In this solution, mitomycin C was dissolved and the pH was adjusted to 8.0 with sodium hydroxide to give a final concentration of 400 μg/ml.

From this solution, a lyophilized injection was prepared using the same procedure as in Example 7.

Example 12) Freeze-dried injection Human EGF was prepared using pyrogen-free 0.001% polyoxyethylene sorbitan fatty acid ester (polysorbate 80).
) to a final concentration of 20μg/
The volume was adjusted to ml. Mitomycin C was dissolved in this solution and adjusted to pH 8.0 with sodium hydroxide to give a final concentration of 400 μg/ml. A lyophilized injection was prepared from this solution using the same procedure as in Example 7.

Example 13) Injection Human EGF 100μg Gelatin 10+ag1- (
2-tetrahydrofuryl) -5-fluorouracil 400mg Trishydroxymethylaminomethane 400mg Distilled water for injection Appropriate amount (pH 9.5 with hydrochloric acid)
(10 ml per ampoule) The solution with the above mixing ratio was sterilized or retired in the same manner as in Example 1, and an injection was prepared.

Example 14) Injection Human EGF i00μg Furil)-5-fluorouracil 400mg Trishydroxymethylaminomethane 400a+g Distilled water for injection Appropriate amount per ampoule
10 ml of the solution having the above-mentioned mixing ratio was filtered for sterilization in the same manner as in Example 1, and then an injection was prepared.

Example 15) Freeze-dried injection A freeze-dried injection was prepared from the injection prepared in Example 13 in the same manner as in Example 7.

Example 16) Freeze-dried injection A freeze-dried injection was prepared from the injection prepared in Example 14 in the same manner as in Example 7.

Example 17 Injection human EGF 100 μg gelatin 5 mg cyclophosphamide (anhydrous) 100 mg sodium chloride
45 mg of distilled water for injection 5 ml per ampoule A solution with the above-mentioned mixing ratio was sterilized and filtered in the same manner as in Example 1, and then an injection was prepared.

Example 18) Injection Human EGF 100 μg Polyoxyethylene sorbitan fatty acid ester (Polysorbate 80) 50 μg Cyclophosphamide (anhydrous) 100 ■ Sodium chloride 45 mg Distilled water for injection
Appropriate amount: 5 ml per ampoule A solution having the above-mentioned mixing ratio was sterilized and filtered in the same manner as in Example 1 to prepare an injection.

Example 19) Freeze-dried injection A freeze-dried injection was prepared from the injection prepared in Example 17 in the same manner as in Example 7.

Example 20) Freeze-dried injection A freeze-dried injection was prepared from the injection prepared in Example 18 in the same manner as in Example 7.

Example 21) Injection Human EGF 100 μg Polyoxyethylene sorbitan fatty acid ester (Polysorbate 80) 500 μg Cisplatin 25 mg Sodium chloride 365 mg Mannitol
500■ Distilled water for injection
50 ml per vial (adjust to pH 2.5 with hydrochloric acid and dissolve human EGF) A solution with the above blending ratio was sterilized and filtered as in Example 1,
Aliquots of 50ml were dispensed into amber glass vials.

The vial was then capped and sealed under aseptic conditions.

Example 22) Freeze-dried injection A sterilization or decommissioning solution with the mixing ratio of Example 21 was added to Example 7.
A lyophilized injection was prepared by dispensing 10 ml of the solution into colored glass vials as follows.

Example 23) Injectable bovine brain FGF was dissolved in pyrogen-free O, 1/15M phosphate buffer containing 1 w/v% gelatin to a final concentration of 2.
The concentration was set to 0 μg/ml. For example, in the case of pH 7.4, sodium chloride was added to a final concentration of 75 mM. This solution was sterilized and filtered in the same manner as in Example 1, and an ampoule of 5 ml was prepared.
After each portion was dispensed, the container was sealed.

Example 24) Injectable bovine brain FGF was dissolved in pyrogen-free l/15M phosphate buffer containing 0.001 w/v% polyoxyethylene sorbitan fatty acid ester (polysorbate 80) to a final concentration of 20 μg/ml. I made it.

For example, in the case of pH 7.4, sodium chloride was added at a final concentration of 75 mM. This solution was sterilized and filtered in the same manner as in Example 1, dispensed into ampoules of 5 ml, and then sealed.

Example 25) Freeze-dried injection A freeze-dried injection was prepared from the injection prepared in Example 23 in the same manner as in Example 3.

Example 26) Freeze-dried injection A freeze-dried injection was prepared from the injection prepared in Example 24 in the same manner as in Example 3.

Example 27) Injection Bovine brain acidic FGF 100μg Gelatin 5rng5-fluorouracil 100mg Trishydroxymethylaminomethane 400mg Distilled water for injection Appropriate amount (pH 8.0 with hydrochloric acid)
(5 ml per ampoule) An injection was prepared by sterilization and filtration in the same manner as in Example 1 using the above mixing ratio.

Example 28) Injection Bovine brain acidic FGF 100 μg Polyoxyethylene sorbitan fatty acid ester (Polysorbate 80) 50 μg 5-fluorouracil 100 mg Trishydroxymethylaminomethane 400 mg Distilled water for injection Appropriate amount (pH 8.0 with hydrochloric acid)
(5 ml per ampoule) An injection was prepared by sterilizing and filtering in the same manner as in Example 1 at the above mixing ratio.

Example 2) Freeze-dried injection A freeze-dried injection was prepared from the injection prepared in Example 27 in the same manner as in Example 7.

Example 30) Freeze-dried injection A freeze-dried injection was prepared from the injection prepared in Example 28 using the same procedure as in Example 7.

Example 31) Injection Contains 0.5w/w% zinc Human insulin 40+, U.

(After dissolving in hydrochloric acid, mix with other ingredients) Sodium acetate 3H201,4II 1g Methyl paraoxybenzoate 1mg Ethyl ester
1■ Anhydrous glycerol
16 ■ Polyoxyethylene sorbitan fatty acid ester (Polysorbet) 80) 10 μg Distilled water for injection Appropriate amount Sodium hydroxide Appropriate amount (adjust to pH 7.4) 1 ml per vial Example 32) Freeze-dried injection 1 0, 5 W/W % zinc-containing human insulin (
7) Dissolved with IN hydrochloric acid. This insulin solution was dissolved in pyrogen-free physiological saline containing 0.0 OIW/V% polyoxyethylene sorbitan fatty acid ester (polysorbate 8o) to a final concentration of 81.0. /ml. Adriamycin hydrochloride was dissolved in this solution to a final concentration of 2 mg/ml. Example 1
It was sterilized and filtered in the same manner.

A lyophilized injection was prepared from the solution using the same procedure as in Example 7.

Example 33) Injection Contains 0.5w/w% zinc Human insulin 401. U.

(After dissolving in hydrochloric acid, mix with other ingredients) Polyoxyethylene sorbitan 250μg Fatty acid ester (polysorbate 80) Cisplatin
25zg sodium chloride
225mg mannitol 250
Distilled water for injection Appropriate amount 25ml per vial (adjust to pH 2.5 with hydrochloric acid) A solution with the above blending ratio was sterilized and filtered as in Example 1,
5ml aliquots were dispensed into 50mf amber glass vials. The vial was then capped and sealed under aseptic conditions.

Example 34) Injection Human EGF 100μg Sodium acetate 6mg Methyl hydroxybenzoate 5mg Anhydrous glycerol
80 ■ Polyoxyethylene sorbitan fatty acid ester (polysorbate 80) 50 μg Distilled water for injection Appropriate amount Sodium hydroxide Appropriate amount (adjust to pH 7, 4) 5 ml per vial Sterile filtration in the same manner as Example 1 with the above mixing ratio An injection was prepared.

Example 35) Injection Human EGF 100 μg Phenol 10 ■ Anhydrous glycerol 80 ■ Polyoxyethylene sorbitan fatty acid ester (polysorbate 110) 50 μg
Distilled water for injection Appropriate amount Sodium hydroxide Appropriate amount (adjusted to pH 7, 4) 5 ml per vial The mixture was sterilized and filtered in the same manner as in Example 1 using the above mixing ratio to prepare an injection.

Example 36) Injection human EGF 100μgm-
Cresol 15mg Anhydrous glycerol 80+ng Polyoxyethylene sorbitan fatty acid ester (Polysorbate 80) 50μg Distilled water for injection Appropriate amount Sodium hydroxide
Appropriate amount (adjusted to pH 7.4) 5 ml per vial The mixture was sterilized and filtered in the same manner as in Example 1 using the above mixing ratio to prepare an injection.

Example 37) Freeze-dried injection Human EGF 50 μg Human EGF II 50 μg Polyoxyethylene sorbitan fatty acid ester (Polysorbet) 80) 50 μg5
-Fluorouracil 1100II1 Trishydroxymethylaminomethane 400■ Appropriate amount of distilled water for injection (adjust to pH 8.0 with hydrochloric acid) 5mlml From the solution sterilized and filtered in the same manner as in Example 1, Example 3. A lyophilized injection was prepared in the same manner.

Example 38) Lyophilized injection human EGF 50 μg human EGF II 50 μg polyoxyethylene sorbitan fatty acid ester (polysorbate 80) 100 μg 1
- (2-Tetrahydrofuryl) -5-Fluorouracil 400mg Trishydroxymethylaminomethane 400ff1g Distilled water for injection Appropriate amount (pH 9 with hydrochloric acid)
, 5) 10 ml A lyophilized injection was prepared in the same manner as in Example 3 from a solution that had been sterilized and filtered in the same manner as in Example 1 at the above mixing ratio.

Example 39) Freeze-dried injection human EGF and human EGFn were prepared in a pyrogen-free 0.0
Dissolved in physiological saline containing 01 w/v% polyoxyethylene sorbitan fatty acid ester (polysorbate 80) to a final concentration of 10 μg/ml (human EGF) and 1
The concentration was adjusted to 0 μz/ml (human EGF■). Dissolve adriamycin hydrochloride in this solution to a final concentration of 2 mg.
/ ml. This solution was sterilized and filtered in the same manner as in Example 1, and a lyophilized injection was prepared from this solution in the same manner as in Example 7.

Example 40) Freeze-dried injections Human EGF and human EGF II were prepared in a pyrogen-free, 0.00-g.
Final concentrations of 10 μg/ml (human EGF) and 10 tt g/ml (human EGF II) were prepared by dissolving in physiological saline containing 0.001 w/v% polyoxyethylene sorbitan fatty acid ester (polysorbate 80). Dissolve mitomycin C in this solution, adjust the pH to 8.0 with sodium hydroxide, and make a final concentration of 400 μg.
/ml.

From this solution, a lyophilized injection was prepared using the same procedure as in Example 7.

Example 41) Injection Human EGF 50 μg Human EGFn 50 μg Polyoxyethylene sorbitan fatty acid ester (Polysorbet) 80) 500 μg Cisplatin 25 ng Sodium chloride 365 mg Mannitol
500 mg distilled water for injection
Appropriate amount 50ml per vial (adjust to pH 2.5 with hydrochloric acid) Sterilize and filter as in Example 1 using the above blending ratio, and add 50m1 per vial.
One portion was dispensed into an amber glass vial.

The vial was then capped and sealed under aseptic conditions.

Example 42) Freeze-dried injection [L eu ”) - Human EGF 10
0 μg 5-fluorouracil 100 mg Trishydroxymethylaminomethane 400 mg Distilled water for injection Appropriate amount (pH 8.0 with hydrochloric acid)
A lyophilized injection was prepared in the same manner as in Example 3 from a solution that had been sterilized and filtered in the same manner as in Example 1 using 5 ml of the above-mentioned mixture.

Example 43) Freeze-dried injection (Leu”) - Human EGF 100 μg
Polyoxyethylene sorbitan fatty acid ester (polysorbate 80) 100μg1
- (Tetrahydrofuryl) - 5-fluorouracil 400 mg Trishydroxymethylaminomethane 400 ■ Distilled water for injection Appropriate amount (adjust to pH 9.5 with hydrochloric acid) 10 ml A solution with the above mixing ratio was sterilized and filtered in the same manner as in Example 1. A lyophilized injection was prepared from the solution in the same manner as in Example 3.

Example 44) Freeze-dried injection (Leu21) - Human EGF was prepared in a pyrogen-free, 0.00-g.
Dissolved in physiological saline containing 001 w/v% polyoxyethylene sorbitan fatty acid ester (polysorbate 80).
The final concentration was adjusted to 20 μg/ml. Dissolve adriamycin hydrochloride in this solution to a final concentration of 2.
mg/ml. This solution was sterilized and filtered in the same manner as in Example 1, and a lyophilized injection was prepared from this solution in the same manner as in Example 7.

Example 45) Freeze-dried injection [Leu21] - Human EGF in pyrogen-free 0.0
It was dissolved in physiological saline containing 01 w/v% polyoxyethylene sorbitan fatty acid ester (polysorbate 80), and the final concentration was adjusted to 20 μg/ml. Dissolve mitomycin C in this solution and ply with sodium hydroxide.
H8,0 was adjusted to a final concentration of 400 μg/ml. From this solution, a lyophilized injection was prepared using the same procedure as in Example 7.

Example 46) Injection [Leu21] - Human EGF 100μ
g Polyoxyethylene sorbitan fatty acid ester (polysorbate 80) 500 μg cisplatin 25 ■ Sodium chloride ° 365 mg mannitol
500mg distilled water for injection
Appropriate amount: 50 ml per vial (adjust to pH 2.5 with hydrochloric acid) A solution with the above mixing ratio was sterilized and filtered in the same manner as in Example 1.
0 ml portions were dispensed into amber glass vials. The vial was then capped and sealed under aseptic conditions.

Example 47) Freeze-dried injection Human TEGFa 100μg Polyoxyethylene sorbitan fatty acid ester (Polysorbate 80) 50μg5
-Fluorouracil 100mg Trishydroxymethylaminomethane 400mg Distilled water for injection Appropriate amount (pH 8.0 with hydrochloric acid)
Example 1 with the above side combination
A lyophilized injection was prepared in the same manner as in Example 3 from a solution that had been sterilized and filtered in the same manner as in Example 3.

Example 48) Freeze-dried injection Human TEGFa 100 μg Polyoxyethylene sorbitan fatty acid ester (Polysorbate 80) 100 μg 1
- (2-Tetrahydrofuryl) -5-Fluorouracil 400mg Trishydroxymethylaminomethane 400mg Distilled water for injection Appropriate amount (pH 9.5 with hydrochloric acid)
10 ml of the above-mentioned solution was filtered for sterilization in the same manner as in Example 1. A lyophilized injection was prepared from this solution in the same manner as in Example 3.

Example 49) Injection Human TGFa 100μg Polyoxyethylene sorbitan fatty acid ester (polysorbate 80) 500μg Cisplatin 25+ng Sodium chloride 365mg Mannitol
500a+g distilled water for injection
Appropriate amount: 50 ml per vial (adjust to pH 2.5 with hydrochloric acid) A solution with the above mixing ratio was sterilized and filtered as in Example 1,
50ml portions were dispensed into amber glass vials. The vial was then capped and sealed under aseptic conditions.

Example 50) Freeze-dried injection Human EGF 100 μg Human TGFβ 50■ Polyoxyethylene sorbitan fatty acid ester (polysorbate 80) 50 μg 5-fluorouracil 100 mg Trishydroxymethylaminomethane 400 mg Distilled water for injection Appropriate amount (pH 8.0 with hydrochloric acid)
) Add 5ml of the above solution to
It was filtered for sterilization in the same manner as in Example 1. From this solution,
A lyophilized injection was prepared in the same manner as in Example 3.

Example 51) Freeze-dried injection human EGF 100μg human TGFβ 501! Ig
Polyoxyethylene sorbitan fatty acid ester (polysorbate 80) 100μg1
- (2-Tetrahydrofuryl) -5-Fluorouracil 400 ■ Trishydroxymethylaminomethane 400 ■ Distilled water for injection Appropriate amount (adjust to pH 9.5 with hydrochloric acid) 10 ml of solution with the above mixing ratio in the same manner as Example 1 Sterilized and filtered. A lyophilized injection was prepared from this solution in the same manner as in Example 3.

Example 52) Freeze-dried injection bovine brain acidic FGF 100 μg polyoxyethylene sorbitan fatty acid ester (polysorbate 80) 100 μg 1
-(2-tetrahydrofuryl) -5-fluorouracil 400 mg Trishydroxymethylaminomethane 400 mg Distilled water for injection Appropriate amount (pH 9 with hydrochloric acid,
5) 10ml Example 1 with the above blending ratio
A lyophilized injection was prepared in the same manner as in Example 3 from a solution that had been filtered for sterilization.

Example 53) Freeze-dried injection 0.5 w/w% zinc-containing human insulin in a minimum amount of I
Dissolved with N-hydrochloric acid. This insulin solution was dissolved in pyrogen-free physiological saline containing 0.001 w/v% polyoxyethylene sorbitan fatty acid ester (polysorbate 80) to a final concentration of 81. The concentration was adjusted to U,/ml. Dissolve mitomycin C in this solution and adjust the pH to 8 with sodium hydroxide to give a final concentration of 400 μg/m
I made it to l.

This solution was sterilized and filtered in the same manner as in Example 1.

From this solution, a lyophilized injection was prepared using the same procedure as in Example 7.

Example 54) Freeze-dried injection Human IGFn 50μg Polyoxyethylene sorbitan fatty acid ester (Polysorbet) 80) 50μg5
-Fluorouracil 100mg Trishydroxymethylaminomethane 400mg Distilled water for injection Appropriate amount (pH 8.0 with hydrochloric acid)
) Add 5ml of the above solution to
It was sterilized and filtered in the same manner as in Example 1. From this solution,
A lyophilized injection was prepared in the same manner as in Example 3.

Example 55) Freeze-dried injection human IGFn 50 μg polyoxyethylene sorbitan fatty acid ester (polysorbate 80) 100 μg 1
- (2-Tetrahydrofuryl) -5-Fluorouracil 400mg Trishydroxymethylaminomethane 400mg Distilled water for injection Appropriate amount (pH 9.5 with hydrochloric acid)
) 10ml of the solution with the above mixing ratio,
Sterilization and filtration were performed in the same manner as in Example 1. From this solution, a lyophilized injection was prepared in the same manner as in Example 3.

Example 56) Freeze-dried injection human IGFII was dissolved in pyrogen-free physiological saline containing 0.001 w/V% polyoxyethylene sorbitan fatty acid ester (polysorbate 80) and 0.0 OIN hydrochloric acid, and the final concentration was 10 μg. /ml. Dissolve adriamycin hydrochloride in this solution to a final concentration of 2.
mg/ml. This solution was sterilized and filtered in the same manner as in Example 1. From this solution, a lyophilized injection was prepared using the same procedure as in Example 7.

Example 57) Freeze-dried injection human IGFm was added to physiological saline containing pyrogen-free 0.001w/V% polyoxyethylene sorbitan fatty acid ester (polysorbate 80) and adjusted to pH 8.0 with sodium hydroxide. to a final concentration of 10 μg.
/ml. In this solution, mitomycin C was dissolved to a final concentration of 400 μg/ml. This solution was sterilized and filtered in the same manner as in Example 1. A lyophilized injection was prepared from this solution in the same manner as in Example 7.

Example 58) Injection human IGFn 50 μg polyoxyethylene sorbitan fatty acid ester (polysorbate 80) 500 μg cisplatin 25 mg sodium chloride 365a+g mannitol
500mg distilled water for injection
Appropriate amount 50 ml per vial (adjust to pH 2.5 with hydrochloric acid and dissolve human IGFII) A solution with the above mixing ratio was sterilized and filtered as in Example 1,
0 ml portions were dispensed into amber glass vials. The vial was then capped and sealed under aseptic conditions.

Example 59) Lyophilized injection human EGF and human EGFn each at a final concentration of 10μ
g/ml, pyrogen-free 0, 1 w/v
% gelatin in 1715M sodium phosphate buffer. The pH was set to 7.4, and sodium chloride was added to a final concentration of 75 mM. This solution was sterilized and filtered in the same manner as in Example 1, and a lyophilized injection was prepared from this solution in the same manner as in Example 3.

Example 60) Freeze-dried injection human EGF and human EGFn each at a final concentration of 10
Pyrogen-free 0.001w so as to be μg/ml
/v% polyoxyethylene sorbitan fatty acid ester (
It was dissolved in 1/15M sodium phosphate buffer containing polysorbate 80). The pH was set to 7.4, and the final concentration was 75 m
M of sodium chloride was added. This solution was removed in the same manner as in Example 1, and a lyophilized injection was prepared from this solution in the same manner as in Example 3.

Example 61) Freeze-dried injection (Leu”)-hEGF in pyrogen-free 0, lv/v
% gelatin in 1715 M sodium phosphate buffer to a final concentration of 20 μg/ml. The pH was set to 7.4, and sodium chloride was added to a final concentration of 75 mM. This solution was sterilized and filtered in the same manner as in Example 1, and a lyophilized injection was prepared from this solution in the same manner as in Example 3.

Example 62) Freeze-dried injection (Leu2')-hEGF in pyrogen-free 0.0
It was dissolved in 1/15M sodium phosphate buffer containing 01 w/v% polyoxyethylene sorbitan fatty acid ester (polysorbate 80) to a final concentration of 20 μg/ml. The pH was set to 7.4, and sodium chloride was added to a final concentration of 75 mM. This solution was filtered to remove bacteria using the same flow as in Example 1, and a lyophilized injection was prepared from this solution in the same manner as in Example 3.

Example 63) Injection of human TGF into a pyrogen-free, 0. It was dissolved in 1715M sodium phosphate buffer containing 1 w/v% Zeαlatin to a final concentration of 20 μg/ml. The pH was set to 5.9, and sodium chloride was added to a final concentration of 89 mM. This solution was sterilized and filtered in the same manner as in Example 1, and ampoule 5
After dispensing ml each, the container was sealed.

Example 64) Injection Human TGFa was dissolved in pyrogen-free 1715M sodium phosphate buffer containing 0.001 v/v% polyoxyethylene sorbicun fatty acid ester (polysorbate 80) to a final concentration of 20 μg/ml. did. pH is 5
．． 9 and added sodium chloride to a final concentration of 89 mM.

This solution was sterilized and filtered in the same manner as in Example 1, dispensed into ampoules of 5 ml, and then sealed.

Example 65) Freeze-dried injection human EGF and human TGFβ were each prepared at a final concentration of 20
pyrogen-free 0.05 μg/ml and 10 mg/ml. It was dissolved in l/15M sodium phosphate buffer containing 1 w/v% gelatin. The pH was set to 7.4,
Sodium chloride was added to a final concentration of 75mM. This solution was sterilized and filtered in the same manner as in Example 1, and a lyophilized injection was prepared from this solution in the same manner as in Example 3.

Example 66) Freeze-dried injection human EGF and human TGFβ were each prepared at a final concentration of 20
l/ml containing 0.001 w/v % pyrogen-free polyoxyethylene sorbitan fatty acid ester (polysorbate 80) to give μg/ml and 10 μg/ml.
Dissolved in 15M sodium phosphate buffer. pH is 7,
4 and added sodium chloride to a final concentration of 75 mM. This solution was sterilized and filtered in the same manner as in Example 1, and a lyophilized injection was prepared from this solution in the same manner as in Example 3.

Example 67) Freeze-dried injection human IGFm was prepared into a pyrogen-free, 0. It was dissolved in 1/15M sodium phosphate buffer containing 1v/V% gelatin to a final concentration of 10 μz/ml. pH is 7.7
Then, sodium chloride was added to a final concentration of 73.6 mM.

This solution was sterilized and filtered in the same manner as in Example 1, and a lyophilized injection was prepared from this solution in the same manner as in Example 3.

Example 68) Lyophilized injection human IGFII was dissolved in pyrogen-free 1/15M sodium phosphate buffer containing 0.001 w/v% polyoxyethylene sorbitan fatty acid ester (polysorbate 80) to adjust the final concentration. The concentration was adjusted to 10 μg/ml. p
H was set to 7.7, and sodium chloride was added at a final concentration of 73.6 mM. This solution was sterilized and filtered in the same manner as in Example 1, and a lyophilized injection was prepared from this solution in the same manner as in Example 3.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the change in gun weight over time in Experimental Example 1. FIG. 2 is a graph showing the change in gun weight over time in Experimental Example 2. FIG. 3 is a graph showing the change in gun weight over time in Experimental Example 3. FIG. 4 is a graph showing changes over time in the body weight of experimental animals in Experimental Example 1. FIG. 5 is a graph showing the change in gun weight over time immediately before compound administration (first white of the eye) in Experimental Example 8. FIG. 6 is a graph showing the change in gun weight over time immediately before compound administration (first white of the eye) in Experimental Example 9. FIG. 7 is a graph showing the change in gun weight over time in Experimental Example 10. FIG. 8 is a graph showing the change in gun weight over time in Experimental Example 13. Applicant's Representative Sato - Number of days after administration of the male compound Figure 6 Number of days after administration of the compound Figure 6 Number of days after administration of the compound

Claims (1)

[Scope of Claims] 1. Contains as active ingredients (a) a compound having an anticancer effect; and (b) a growth factor, a peptide corresponding to a part of its constituent components, a derivative thereof, or a salt thereof. An anticancer agent characterized by: 2. The anticancer agent according to claim 1, wherein component (a) is an alkylating agent, an antimetabolite, an antibiotic, or a platinum preparation. 3. The anticancer agent according to claim 2, wherein component (a) is an alkylating agent. 4. The anticancer agent according to claim 3, wherein the alkylating agent is cyclophosphamide, a derivative thereof, or a salt thereof. 5. The anticancer agent according to claim 2, wherein component (a) is an antimetabolite. 6. The anticancer agent according to claim 5, wherein the antimetabolite is 5-fluorouracil, tegafur, a derivative thereof, or a salt thereof. 7. Claim 2 in which component (a) is an antibiotic
Anticancer drugs listed in section. 8. The anticancer agent according to claim 7, wherein the antibiotic is mitomycin, a derivative thereof, or a salt thereof. 9. Claim 2, in which component (a) is a platinum preparation
Anticancer drugs listed in section. 10. The anticancer agent according to claim 9, wherein the platinum preparation is cisplatin. 11. The anticancer agent according to any one of claims 1 to 10, wherein component (b) is an epidermal growth factor family member, an insulin family member, or a platelet-derived growth factor family member. 12. The anticancer agent according to claim 11, wherein component (b) is an epidermal growth factor family member. 13. The epidermal growth factor family includes human epidermal growth factor (
hEGF), N among the 53 amino acid residues that constitute hEGF
One in which the 21st methionine from the end is replaced with leucine ([Leu^2^1]-hEGF), and one in which two of the 53 amino acid residues that make up hEGF, leucine and arginine, are missing from the C-terminus (hEGF-II ), α-form transforming growth factor (TGF_α), or β-form transforming growth factor (TGF_β),
The anticancer agent according to claim 12. 14. The anticancer agent according to claim 11, wherein component (b) is an insulin family member. 15. The insulin family includes insulin, insulin-like growth factor type I (IGF-I), or insulin-like growth factor
The anticancer agent according to claim 14, which is type II (IGF-II). 16. The anticancer agent according to claim 11, wherein component (b) is a platelet-derived growth factor group. 17. The platelet-derived growth factor family is fibroblast growth factor (F
The anticancer agent according to claim 16, which is GF).